Methods and compositions for inhibiting the growth of hematopoietic malignant cells

ABSTRACT

Disclosed herein are compositions and methods for reducing the growth of hematopoietic malignant cells (e.g., B-cell leukemia cells). The methods involve reducing the growth of hematopoietic malignant cells by contacting hematopoietic malignant cells with GP88 antagonists. GP88 is an 88 KDa autocrine growth factor that promotes the growth of hematopoictic malignant cells. Antagonists to GP88 are provided which inhibit its expression or biological activity. The antagonists include antisense oligonucleotides and antibodies. Also provided are methods for determining if a patient is responding or is responsive to anti-cancer therapy (e.g., glucocorticoid therapy). Increased levels of GP88 in hematopoietic cells indicates a patient is not responding or responsive to anti-cancer therapy.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/456,886, filed Dec. 8, 1999, which is a divisional of U.S.application Ser. No. 08/991,862, filed Dec. 16, 1997, now U.S. Pat. No.6,309,826, which is a continuation-in-part of U.S. patent applicationSer. No. 08/863,079, filed May 23, 1997, now abandoned.

FIELD OF THE INVENTION

[0002] This invention relates to cell biology, physiology and medicine,and concerns an 88 kDa glycoprotein growth factor (“GP88” or “PCDGF”)and compositions and methods which affect the expression and biologicalactivity of GP88. These compositions and methods are useful fordiagnosis and treatment of diseases including cancer.

REFERENCES

[0003] Several publications are referenced herein by Arabic numeralswithin parenthesis. Full citations for these references may be found atthe end of the specification immediately preceding the claims.

BACKGROUND OF THE INVENTION

[0004] The proliferation and differentiation of cells in multicellularorganisms is subject to a highly regulated process. A distinguishingfeature of cancer cells is the absence of control over this process;proliferation and differentiation become deregulated resulting inuncontrolled growth. Significant research efforts have been directedtoward better understanding this difference between normal and tumorcells. One area of research focus is growth factors and, morespecifically, autocrine growth stimulation.

[0005] Growth factors are polypeptides which carry messages to cellsconcerning growth, differentiation, migration and gene expression.Typically, growth factors are produced in one cell and act on anothercell to stimulate proliferation. However, certain malignant cells, inculture, demonstrate a greater or absolute reliance on an autocrinegrowth mechanism. Malignant cells which observe this autocrine behaviorcircumvent the regulation of growth factor production by other cells andare therefore unregulated in their growth.

[0006] Study of autocrine growth control advances understanding of cellgrowth mechanisms and leads to important advances in the diagnosis andtreatment of cancer. Toward this end, a number of growth factors havebeen studied, including insulin-like growth factors (“IGF-I” and“IGF-II”), gastrin-releasing peptide (“GRP”), transforming growthfactors alpha and beta (“TGF-a” and “TGF-b”), and epidermal growthfactor (“EGF”).

[0007] The present invention is directed to a recently discovered growthfactor. This growth factor was first discovered in the culture medium ofa highly tumorigenic “PC cell line,” an insulin-independent variantisolated from the teratoma derived adipogenic cell line 1246. Thisgrowth factor is referred to herein as “GP88.” GP88 has been purifiedand structurally characterized. Amino acid sequencing of GP88 indicatesthat GP88 has amino acid sequence similarities with the mousegranulin/epithelin precursor.

[0008] Granulins/epithelins (“grn/epi”) are 6 kDa polypeptides andbelong to a novel family of double cysteine rich polypeptides. U.S. Pat.No. 5,416,192 (Shoyab et al.) is directed to 6 kDa epithelins,particularly epithelin 1 and epithelin 2. According to Shoyab, bothepithelins are encoded by a common 63.5 kDa precursor, which isprocessed into smaller forms as soon as it is synthesized, so that theonly natural products found in biological samples are the 6 kDa forms.Shoyab et al. teaches that the epithelin precursor is biologicallyinactive.

[0009] Contrary to the teachings of Shoyab et al., the inventor'slaboratory has demonstrated that the precursor is not processed as soonas it is synthesized. Studies, conducted in part by this inventor, havedemonstrated that the precursor (i.e., GP88) is in fact secreted as an88 kDa glycoprotein with an N-linked carbohydrate moiety of 20 kDa.Analysis of the N-terminal sequence of GP88 indicates that GP88 startsat amino acid 17 of the grn/epi precursor, demonstrating that the first17 amino acids from the protein sequence deduced from the precursor cDNAcorrespond to a signal peptide compatible with targeting for membranelocalization or for secretion.

[0010] Also in contrast to the teachings of Shoyab et al., the inventordemonstrated that GP88 is biologically active and has growth promotingactivity, particularly as an autocrine growth factor for the producercells.

[0011] Hematopoietic malignancies are malignant blood diseases includingvarious lymphomas and leukemias. Leukemias of B-cell lineage include,but are not limited to, acute lymphocytic leukemia, B cell lymphoma, andmultiple myeloma. Multiple myeloma (“MM”) is a clonal B-cell neoplasmand the second most prevalent blood cancer, representing 1% of allcancers and 2% of all cancer deaths. B-cells (or B-lymphocytes) areprecursor cells that differentiate into plasma cells after exposure toparticular antigens. Plasma cells produce immunoglobulins and have alimited life span. However, uncontrolled growth of plasma cells in aclonal lineage of B cells may lead to accumulation of plasma cellsproducing monoclonal immunoglobulins or immunoglobulin fragments (e.g.,M protein). MM is characterized by bone degradation and fractures,anemia, increased risk of infection, and decreased production ofplatelets in addition to other symptoms. The incidence of MM, currentlyabout 14,000 new cases per year, has been steadily increasing in theUnited States for several decades (1). There has been little improvementin the treatment of human MM over the past 25 years and there is no curefor the disease (3). The few available therapies for treatment of MMhave severe side effects and are of limited efficacy. For nearly 3decades, the standard treatment for human MM has been glucocorticoidand/or chemotherapy with melphalan and prednisone alone or combinationsof alkylating agents such as glucocorticoids and anthracyclines (4).However, almost all patients with MM who initially respond toglucocorticoid therapy relapse, with a median survival of two to threeyears following diagnosis (5). During the progression of MM to moreaggressive forms of the disease, MM cells become insensitive to thekilling effect of glucocorticoids leaving only the use ofchemotherapeutic agents to control the disease.

[0012] What is needed are new compositions and methods for treatment anddiagnosis of MM, and particularly compositions and methods that inhibitthe proliferation and survival of multiple myeloma cells.

SUMMARY OF INVENTION

[0013] The inventor has now unexpectedly discovered that a glycoprotein(GP88), which is expressed in a tightly regulated fashion in normalcells, is overexpressed and unregulated in highly tumorigenic cellsderived from the normal cells, that GP88 acts as a stringently requiredgrowth stimulator and survival factor for the tumorigenic cells and thatinhibition of GP88 expression or action in the tumorigenic cells resultsin an inhibition of the tumorigenic properties of the overproducingcells.

[0014] The inventor has further discovered that GP88 is overexpressed inhematopoietic malignant cells such as leukemia cells of B-cell lineage(e.g., acute lymphocytic leukemia, B cell lymphoma, and multiplemyeloma). GP88 stimulates the tumorigenic properties of hematopoieticmalignant cells while inhibition of GP88 expression and biologicalactivity greatly reduces the tumorigenic properties of hematopoieticmalignant cells. An embodiment of the invention provides methods ofinhibiting the growth or viability of hematopoietic malignant cells. Inone embodiment of the invention, a GP88 antagonist inhibits multiplemyeloma cell growth. In another embodiment of the invention, acomposition for inhibiting the growth or viability of hematopoieticmalignant cells comprising a GP88 antagonist (e.g., an anti-GP88antibody, or anti-GP88 nucleic acid) is provided. In yet anotherembodiment, a method of diagnosing B-cell leukemia is providedcomprising detecting GP88 (e.g., GP88 protein, or nucleic acids encodingGP88) in a tissue sample containing B cells (e.g., tissue suspected ofcontaining myeloma cells including, but not limited to blood, bonemarrow, lymph, liver, and spleen) and diagnosing multiple myeloma bydetermining whether GP88 protein is present in the tissue sample. Thepresence of GP88 in B cells indicates multiple myeloma. Alternatively,detecting GP88 in B-cells indicates the presence of leukemia cells ofB-cell lineage. Thus, the presence of GP88 serves as a prognostic markerfor B-cell leukemia.

[0015] The invention also provides methods for determining whether apatient is responding or responsive to glucocorticoid therapy bycomparing the level of GP88 in a tissue sample containing B-cells at afirst time with the level of GP88 in a tissue sample containing B-cellsat a second time. Increased levels of GP88 in tissue samples over timeindicate a patient is not responding or responsive to glucocorticoidtherapy.

[0016] This invention provides GP88 antagonizing compositions capable ofinhibiting the expression or activity of GP88, methods for treatingdiseases associated with a defect in GP88 quantity or activity such asbut not limited to cancer in a mammal in tissues including, for example,blood, cerebrospinal fluid, serum, plasma, urine, nipple aspirate,liver, kidney, breast, bone, bone marrow, testes, brain, ovary, skin,and lung, methods for determining the susceptibility of a subject todiseases associated with a defect in GP88 expression or action, methodsfor measuring susceptibility to GP88 antagonizing therapy, and methods,reagents, and kits for the in vitro and in vivo detection of GP88 andtumorigenic activity in cells.

[0017] Additional objects and advantages of the invention will be setforth in part in the description that follows, and in part will beobvious from the description, or may be learned by the practice of theinvention.

[0018] To achieve the objects and in accordance with the purpose of theinvention, as embodied and properly described herein, the presentinvention provides compositions for diagnosis and treatment of diseasessuch as but not limited to multiple myeloma in which cells exhibit analtered expression of GP88 or altered response to GP88.

[0019] Use of the term “altered expression” herein means increasedexpression or overexpression of GP88 by a statistically significantamount as compared to corresponding normal cells or surroundingperipheral cells. The term “altered expression” also means expressionwhich became unregulated or constitutive without being necessarilyelevated. Use of the terms increased or altered “response” to GP88 meansa condition wherein increase in any of the biological functions (e.g.,growth, differentiation, viral infectivity) conferred by GP88 results inthe same or equivalent condition as altered expression of GP88.

[0020] Use of the term “GP88” herein means epithelin/granulin precursorin cell extracts and extracellular fluids, and is intended to includenot only GP88 according to the amino acid sequences included in FIG. 8or 9, which are of mouse and human origins, but also GP88 of otherspecies. “GP88” does not include epithelin 1 or epithelin 2 peptides asdescribed in U.S. Pat. No. 5,416,192 (Shoyab et al.). In addition, theterm also includes functional derivatives thereof having additionalcomponents such as a carbohydrate moiety including a glycoprotein orother modified structures.

[0021] Also intended by the term GP88 is any polypeptide fragment havingat least 10 amino acids present in the above mentioned sequences.Sequences of this length are useful as antigens and for maltingimmunogenic conjugates with carriers for the production of antibodiesspecific for various epitopes of the entire protein. Such polypeptidesare useful in screening such antibodies and in the methods directed todetection of GP88 in biological fluids. It is well known in the art thatpeptides are useful in generation of antibodies to larger proteins (7).In one embodiment of this invention, it is shown that peptides from12-19 amino-acids in length have been successfully used to developantibodies that recognize full length GP88.

[0022] The polypeptide of this invention may exist covalently ornon-covalently bound to another molecule. For example, it may be fusedto one or more other polypeptides via one or more peptide bonds such asglutathione transferase, poly-histidine, or myc tag.

[0023] The polypeptide is sufficiently large to comprise anantigenetically distinct determinant or epitope which can be used as animmunogen to reproduce or test antibodies against GP88 or a functionalderivative thereof.

[0024] One embodiment includes the polypeptide substantially free ofother mammalian peptides. GP88 of the present invention can bebiochemically or immunochemically purified from cells, tissues or abiological fluid. Alternatively, the polypeptide can be produced byrecombinant means in a prokaryotic or eukaryotic expression system andhost cells.

[0025] “Substantially free of other mammalian polypeptides” reflects thefact that the polypeptide can be synthesized in a prokaryotic or anon-mammalian or mammalian eukaryotic organism, if desired.Alternatively, methods are well known for the synthesis of polypeptidesof desired sequences by chemical synthesis on solid phase supports andtheir subsequent separation from the support. Alternatively, the proteincan be purified from tissues or fluids of mammals where it naturallyoccurs so that it is at least 90% pure (on a weight basis) or even 99%pure, if desired, of other mammalian polypeptides, and is thereforesubstantially free from them. This can be achieved by subjecting thetissue extracts or fluids to standard protein purification such as onimmunoabsorbants bearing antibodies reactive against the protein. Oneembodiment of the present invention describes purification methods forthe purification of naturally occurring GP88 and of recombinant GP88expressed in baculovirus infected insect cells. Alternatively,purification from such tissues or fluids can be achieved by acombination of standard methods such as but not limited to the onesdescribed in reference (4).

[0026] As an alternative to a native purified or recombinantglycoprotein or polypeptide, “GP88” is intended to also includefunctional derivatives. By functional derivative is meant a “fragment,”“variant,” “analog,” or “chemical derivative” of the protein orglycoprotein as defined below. A functional derivative retains at leasta portion of the function of the full length GP88 which permits itsutility in accordance with the present invention.

[0027] A “fragment” of GP88 refers to any subset of the molecule that isa shorter peptide that retains the tumorigenic properties of thefull-length GP88 protein. This corresponds for example but is notlimited to regions such as K19T and S14R for mouse GP88, and E19V andA14R (equivalent to murine K19T and S14R, respectively) for human GP88.

[0028] A “variant” of GP88 refers to a molecule substantially similar toeither the entire peptide or a fragment thereof. Variant peptides may beprepared by direct chemical synthesis of the variant peptide usingmethods known in the art.

[0029] Alternatively, amino acid sequence variants of the peptide can beprepared by modifying the DNA which encodes the synthesized protein orpeptide. Such variants include, for example, deletions, insertions, orsubstitutions of residues within the amino-acid sequence of GP88. Anycombination of deletion, insertion, and substitution may also be made toarrive at the final construct, provided the final construct possessesthe desired activity. The mutation that will be made in the DNA encodingthe variant peptide must not alter the reading frame and preferably willnot create complementary regions that could produce secondary mRNAstructures. At the genetic level these variants are prepared by sitedirected mutagenesis (8) of nucleotides in the DNA encoding the peptidemolecule thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. The variant typicallyexhibits the same qualitative biological activity as the nonvariantpeptide.

[0030] An “analog” of GP88 protein refers to a non-natural moleculesubstantially similar to either the entire molecule or a fragmentthereof.

[0031] A “chemical derivative” contains additional chemical moieties notnormally a part of the peptide or protein. Covalent modifications of thepeptide are also included within the scope of this invention. Suchmodifications may be introduced into the molecule by reacting targetedamino-acid residues of the peptide with an organic derivatizing agentthat is capable of reacting with selected side chains or terminalamino-acid residues. Most commonly derivatized residues are cysteinyl,histidyl, lysinyl, arginyl, tyrosyl, glutaminyl, asparaginyl and aminoterminal residues. Hydroxylation of proline and lysine, phosphorylationof hydroxyl groups of seryl and threonyl residues, methylation of thealpha-amino groups of lysine, histidine, and histidine side chains,acetylation of the N-terminal amine and amidation of the C-terminalcarboxylic groups. Such derivatized moieties may improve the solubility,absorption, biological half life and the like. The moieties may alsoeliminate or attenuate any undesirable side effect of the protein andthe like. In addition, derivatization with bifunctional agents is usefulfor cross-linking the peptide to water insoluble support matrices or toother macromolecular carriers. Commonly used cross-linking agentsinclude glutaraldehyde, N-hydroxysuccinimide esters, homobifunctionalimidoesters, 1,1-bis(-diazoloacetyl)-2-phenylethane, and bifunctionalmaleimides. Derivatizing agents such asmethyl-3-[9p-azidophenyl)]dithiopropioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287 and 3,691,016 may be employed for proteinimmobilization.

[0032] Use of the term GP88 “antagonizing agents” herein means anycomposition that inhibits or blocks GP88 expression, production orsecretion, or any composition that inhibits or blocks the biologicalactivity of GP88. This can be achieved by any mode of action such as butnot limited to the following:

[0033] (A) GP88 antagonizing agents include any reagent or moleculeinhibiting GP88 expression or production including but not limited to:

[0034] (1) antisense GP88 DNA or RNA molecules that inhibit GP88expression by inhibiting GP88 translation;

[0035] (2) reagents (hormones, growth factors, small molecules) thatinhibit GP88 mRNA and/or protein expression at the transcriptional,translational or post-transaltional levels;

[0036] (3) factors, reagents or hormones that inhibit GP88 secretion;

[0037] (B) GP88 antagonizing agents also include any reagent or moleculethat will inhibit GP88 action or biological activity such as but notlimited to:

[0038] (1) neutralizing antibodies to GP88 that bind the protein andprevent it from exerting its biological activity;

[0039] (2) antibodies to the GP88 receptor that prevent GP88 frombinding to its receptor and from exerting its biological activity;

[0040] (3) competitive inhibitors of GP88 binding to its receptors(e.g., proteins, ribozymes, aptamers, small molecules);

[0041] (4) inhibitors of GP88 signaling pathways (e.g., proteins,ribozymes, aptamers, small molecules).

[0042] Specific examples presented herein provide a description ofpreferred embodiments, particularly the use of neutralizing antibodiesto inhibit GP88 biological action and the growth of multiple myclomacells; the use of antisense GP88 cDNA and antisense GP88oligonucleotides to inhibit GP88 expression leading to inhibition of thetumorigenic properties of PC cells; characterization of GP88 receptorson cell surfaces of several cell lines including the mammary epithelialcell line C57MG, the 1246 and PC cell lines and the mink lung epithelialcell line CCL64.

[0043] In one embodiment of the invention, the GP88 antagonizing agentsare antisense oligonucleotides to GP88. The antisense oligonucleotidespreferably inhibit GP88 expression by inhibiting translation of the GP88protein. In another embodiment, the antagonizing agent is RNAi (RNAinterference nucleic acids). RNAi are double-stranded RNA molecules thatare homologous to the target gene (e.g., GP88) and interfere with thetarget gene's activity.

[0044] Alternatively, such a composition may comprise reagents, factorsor hormones that inhibit GP88 expression by regulating GP88 genetranscriptional activity. Such a composition may comprise reagents,factors or hormones that inhibit GP88 post-translational modificationand its secretion. Such a composition may comprise reagents that act asGP88 antagonists that block GP88 activity by competing with GP88 forbinding to GP88 cell surface receptors. Alternatively, such acomposition may comprise factors or reagents that inhibit the signalingpathway transduced by GP88 once binding to its receptors on diseasedcells.

[0045] The composition may also comprise reagents that block GP88 actionsuch as an antibody specific to GP88 that neutralizes its biologicalactivity, or an antibody to the GP88 receptor that blocks its activity.

[0046] The antibodies of the invention (neutralizing and others) arepreferably used as a treatment for multiple myeloma or other diseases incells which exhibit an increased expression of GP88. By the term“neutralizing” it shall be understood that the antibody has the abilityto inhibit or block the normal biological activity of GP88, includingGP88's ability to stimulate cell proliferation, increase cell survival,or to induce tumor growth in experimental animals and in humans. Aneffective amount of anti-GP88 antibody is administered to an animal,including humans, by various routes. In an alternative embodiment, theanti-GP88 antibody is used as a diagnostic to detect cells which exhibitan altered (increased) expression of GP88 as occurring in diseases suchas but not limited to cancers (e.g., multiple mycloma), and to identifydiseased cells whose growth is dependent on GP88 and which will respondto GP88 antagonizing therapy. In yet another embodiment, the anti-GP88antibody is used to deliver compounds such as cytotoxic factors orantisense oligonucleotides to cells expressing or responsive to GP88.The cytotoxic factors may be attached, linked, or associated with theanti-GP88 antibody.

[0047] The antisense oligonucleotides of the invention are also used asa treatment for cancer in cells which exhibit an increased expression ofGP88, such as hematopoietic malignant cells (e.g., B-cell leukemiacells). An effective amount of the antisense oligonucleotide isadministered to an animal, including humans, by various routes.

[0048] The present invention also provides a method for determining thesusceptibility to diseases associated with a defect in GP88 expressionor action which comprises obtaining a sample of biological fluid ortissue and measuring the amount of GP88 in the fluid or tissue ormeasuring the susceptibility of the cells to respond to GP88. In thecase of cancer (e.g., hematopoietic malignancy), the amount of GP88being proportional to the susceptibility to the cancer.

[0049] The present invention also provides a method for measuring thedegree of severity of cancer (e.g., hematopoietic malignancy) whichcomprises obtaining a sample of biological fluid or tissue and measuringthe amount of GP88 in the fluid or tissue sample, the amount of GP88being proportional to the degree or severity of the cancer. In oneembodiment of the invention, the tissue sample is derived from bone,bone marrow, or serum. In another embodiment of the invention, thepresence of GP88 in B cells is detected.

[0050] The present invention also provides a method for measuringsusceptibility to GP88 antagonizing therapy which comprises obtaining asample of the diseased tissue (biopsy) or a tissue suspected of beingdiseased, maintaining the cells derived from the sample in culture,treating the cells derived from the culture with anti-GP88 neutralizingantibody and determining if the neutralizing antibody inhibits the cellgrowth. The ability of the antibody to inhibit cell growth is indicativethat the cells are dependent on GP88 to proliferate and is predictivethat GP88 antagonizing therapy will be efficacious. In addition, theinvention provides methods for determining whether a patient isresponding or responsive to glucocorticoid therapy by comparing thelevel of GP88 in a tissue sample takent at a first time with a tissuesample taken at a second time. Increased levels of GP88 in tissuesamples containing B-cells indicates the patient is not responding or isnot responsive to glucocorticoid therapy.

[0051] The present invention also provides a method for determining thesusceptibility to cancer associated with an abnormality in GP88 receptorlevel or activity which comprises obtaining a sample of tissue andmeasuring the amount of GP88 receptor protein or mRNA in the tissue ormeasuring the kinase activity of the receptor in the tissue (GP88binding to its receptor induces phosphorylation of cellular proteinsincluding the receptor for GP88).

[0052] The present invention also provides a method for targeting GP88antagonizing reagents to the diseased site by conjugating them to ananti-GP88 antibody or an anti-GP88 receptor antibody.

[0053] The accompanying drawings, which are incorporated in andconstitute a part of this specification, illustrate embodiments of theinvention and, together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0054]FIG. 1A compares the level of expression of GP88 protein in the1246, 1246-3A and PC cell lines. Cells were cultured in DME-F12 mediumsupplemented with 2% fetal bovine serum (FBS). Immunoprecipitation andWestern blot analysis with anti-K19T antibody measured GP88 expressionlevels.

[0055]FIG. 1B compares the level of GP88 mRNA expression in the 1246,1246-3A and PC cell lines. mRNA for RPL32 is used as an internal controlfor equal amounts of RNA loading.

[0056]FIG. 1C compares the expression of GP88 mRNA in 1246 cells (leftpanel) and in PC cells (right panel) in serum-free and serum containingmedium. The results show that GP88 expression in 1246 cells is inhibitedby the addition of fetal bovine serum whereas such inhibition is notobserved in the highly tumorigenic PC cells.

[0057]FIG. 2 illustrates the effect of treatment of the highlytumorigenic PC cells with increasing concentrations of anti-GP88neutralizing antibody.

[0058]FIG. 3 shows C3H mice injected subcutaneously with 106 antisenseGP88 transfected PC cells (bottom) and with empty vector transfectedcontrol PC cells (top).

[0059]FIG. 4 shows in vivo GP88 expression levels in C3H mice tumortissues and in surrounding normal tissues.

[0060]FIG. 5 shows GP88 mRNA expression levels in estrogen receptorpositive and estrogen receptor negative human mammary carcinoma celllines.

[0061]FIG. 6 shows the effect of increasing concentrations of GP88 onthe growth of the mouse mammary epithelial cell line C57.

[0062]FIG. 7 shows the growth properties and tumorigenic ability of PCcells transfected with a cytomegalovirus promoter controlled expressionvector containing GP88 in antisense orientation and PC cells transfectedwith an empty vector.

[0063]FIG. 8 shows the nucleotide and deduced amino-acid sequence ofmouse GP88. Peptide regions used as antigens to raise anti-GP88antibodies K19T and S14R are underlined. The region cloned in theantisense orientation in the pCMV4 mammalian expression vector isindicated between brackets.

[0064]FIG. 9A shows the nucleotide sequence of human GP88 cDNA.Indicated between brackets is the region cloned in the antisenseorientation into the pcDNA3 mammalian expression system; and

[0065]FIG. 9B shows the deduced amino-acid sequence of human GP88. TheE19V region used as antigen to develop anti-human GP88 neutralizingantibody is underlined. It also indicates the region K14R equivalent tothe mouse S14R region.

[0066]FIG. 10 shows the amino-acid sequence of mouse GP88 arranged toshow the 7 and one-half repeats defined as granulins g, f, B, A, C, Dand e (right side). This representation shows that the region K19T andS14R used to raise GP88 antibodies for developing anti-GP88 neutralizingantibodies is found between two epithlin/granulin repeats in what isconsidered a variant region. Indicated on the right hand side is thegranulin classification of the repeats according to Bateman et al (6).Granulin B and granulin A are also defined as epithelin 2 and epithelin1 respectively according to Plowman et al., 1992 (5).

[0067]FIG. 11 shows a schematic representation of pCMV4 and a GP88 cDNAclone indicating the restriction sites used to clone GP88 antisense cDNAinto the expression vector.

[0068]FIG. 12 shows the cross-linking of ¹²⁵I-rGP88 to GP88 cell surfacereceptors on CCL-64 cells. The cross-linking reaction was carried outwith disuccinimidyl suberate (DSS). Reaction products were analyzed bySDS-PAGE on a 7% polyacrylamide gel.

[0069]FIG. 13 shows the cross-linking of ¹²⁵1-rGP88 to GP88 cell surfacereceptors on 3T3 fibroblasts, PC cells and C57MG mammary epithelialcells. The results show that these various cell lines display GP88 cellsurface receptors of similar molecular weight as the ones on CCL64 cells(FIG. 12).

[0070]FIG. 14 shows GP88 expression levels in non-tumorigenic MCF 10Aand in malignant (MCF 7, MDA-468) human mammary epithelial cells.

[0071]FIG. 15 shows that GP88 expression is inhibited by antisense GP88cDNA transfection in human breast carcinoma MDA-468 cells.

[0072]FIG. 16 shows GP88 protein expression in various humanhematological cell lines. GP88 is expressed in human multiple myelomacell lines ARP-1 and RPMI 8226, human B cell lines Raji and Daudi, humanmacrophage cell line KOPM28, but not in human T cell lines Jurkat andKOPT-K1.

[0073]FIG. 17 shows that GP88 mRNA is expressed in human multiplemyeloma cell lines ARP-1 and RPMI 8226.

[0074]FIGS. 18A and 18B show the effect of GP88 protein on the growth ofRPMI 8226 cells. FIG. 18A shows that GP88 increases the live celldensity of serum starved RPMI 8226 cells while FIG. 18B shows that GP88increases the percent viability of serum starved RPMI 8226.

[0075]FIGS. 19A and 19B show the effect of GP88 on the growth of ARP-1cells. FIG. 18A shows that GP88 increases the live cell density of serumstarved ARP-1 cells while FIG. 18B shows that GP88 increases the percentviability of serum starved ARP-1 cells.

[0076]FIG. 20 shows the effect of anti-GP88 neutralizing antibody on thegrowth of RPMI 8226 cells. Treatment of RPMI 8226 cells with anti-GP88antibody inhibited cell growth by 50% compared to cells that did notreceive GP88 antibody (control AB) or cells treated with a combinationof GP88 and anti-GP88 antibody.

[0077]FIGS. 21A and 21B show the effect of GP88 and PD98059 (a MEKinhibitor) on cell growth and survival. GP88 increased both live celldensity (FIG. 21A) and percent survival (FIG. 21B) in ARP-1 cells. Theseresults show that GP88 activates the MAPK pathway in ARP-1 cells andthat MAPK stimulates GP88-induced cell growth.

[0078]FIG. 22 shows that phosphorylation of Erk1 and Erk2 through theMAPK pathway is blocked by MEK inhibitor PD98059 in ARP-1 cells. Theseresults show that GP88 activates the MAPK pathway in ARP-1 cells andthat MAPK stimulates GP88-induced cell growth.

[0079]FIG. 23 shows that GP88 stimulated phosphorylation of Ak1 in ARP-1cells is blocked by PI3 K inhibitor LY294002. The results show that GP88activates the PI3 kinase signal pathway in ARP-1 cells.

[0080]FIG. 24 shows that GP88 does not induce phosphorylation of STAT3.The results show that GP88 does not activate the JAK/STAT3 signalpathway in human multiple myeloma cells.

[0081]FIGS. 25A, 25B, and 25C show the results of triple-stained bonemarrow smears from multiple myeloma patients. The bone marrow smearswere stained for the presence of GP88 and for markers of the kappa andlambda light chains. The bone marrow smears were stained with DAPI (FIG.25A), anti-human kappa/lambda chain antibody (FIG. 25B), and anti-GP88antibody (25C).

[0082]FIG. 26 shows that the effect of dexamethasone on the expressionof GP88 mRNA in multiple myeloma (ARP-1) cells. Dexamethasonesignificantly inhibits the expression of GP88 mRNA in ARP-1 cells.

[0083]FIGS. 27A and 27B shows the effects of GP88 on the cell growth(27A) and viability (27B) of dexamethasone-treated ARP-1 cells.Dexamethasone decreases the cell growth and viability of ARP-1 cells.GP88 partially reverses the negative effects of dexamethasone on thecell growth and viability of ARP-1 cells.

[0084]FIG. 28 shows the effect of GP88 on PARP cleavage indexamethasone-treated ARP-1 cells. GP88 significantly reduces PARPcleavage at 24 and 48 hours following treatment with dexamethasone.

[0085]FIG. 29 shows GP88 protein expression in ARP-1 cells transfectedwith GP88 nucleic acid (lane 2) and an empty vector that does notcontain GP88 nucleic acid (lane 1). GP88 is overexpressed in ARP-1 cellstransfected with GP88 nucleic acid.

[0086]FIGS. 30A and 30B shows the effect of dexamethasone on the cellgrowth (30A) and viability (30B) of ARP-1 cells transfected with GP88nucleic acid and ARP-I cells transfected with an empty vector. Thedecrease in cell growth and viability of ARP-1 cells treated withdexamethasone is significantly reduced in cells transfected with GP88nucleic acid.

[0087]FIG. 31 shows the effect of dexamethasone on PARP cleavage inARP-1 cells overexpressing GP88. ARP-1 cells overexpressing GP88 havesignificantly reduced levels of PARP cleavage.

DETAILED DESCRIPTION OF THE INVENTION

[0088] Reference will now be made in detail to the presently preferredembodiments of the invention, which, together with the followingexamples, serve to explain the principles of the invention.

Biological Activity of GP88

[0089] The invention relates to GP88 and antitumor compositions usefulfor treating and diagnosing diseases linked to altered (increased)expression of GP88 (e.g., multiple myeloma). Alternatively thisinvention is used for treating and diagnosing diseases linked toincreased responsiveness to GP88. Using a murine model system consistingof three cell lines, the inventor has shown that cells which overexpressGP88 form tumors. The parent cell line, 1246, is a C3H mouse adipogeniccell line which proliferates and differentiates into adipocytes in adefined medium under stringent regulation by insulin. The 1246 cellscannot form tumors in a syngeneic animal (C3H mouse) even when injectedat a high cell density. An insulin independent cell line, 1246-3A, wasisolated from 1246 cells maintained in insulin-free medium. The 1246-3Acells lost the ability to differentiate and form tumors when 106 areinjected subcutaneously in syngeneic mice. A highly tumorigenic cellline, PC, was developed from 1246-3A cells by an in vitro-in vivoshuttle technique. The PC cells formed tumors when 104 cells wereinjected into syngeneic mice.

[0090] GP88 is overexpressed in the insulin-independent tumorigenic celllines relative to the parent non-tumorigenic insulin-dependent cellline. Moreover, the degree of overexpression of GP88 positivelycorrelates with the degree of tumorigenicity of these cells,demonstrating for the first time that GP88 is important in tumorigenesis(FIG. 1). With reference to FIG. 1, since GP88 is synthesized by cellsbut also secreted in culture medium, the level of GP88 was determined incell lysates and in culture medium (CM). All cells were cultivated inDME/F12 nutrient medium supplemented with 2% fetal bovine serum. Whencells reached confluency, culture medium (CM) was collected and celllysates were prepared by incubation in buffer containing detergentfollowed by a 10,000×g centrifugation. Cell lysate and conditionedmedium were normalized by cell number. Samples from cell lysate andconditioned medium were analyzed by Western blot analysis using ananti-GP88 antibody, as explained below.

[0091] The development of a neutralizing antibody confirmed GP88's keyrole in tumorigenesis. When an anti-GP88 antibody directed to the K19Tregion of mouse GP88 was added to the culture medium, the growth ofhighly tumorigenic PC cells was inhibited in a dose dependent fashion(FIG. 2). With reference to FIG. 2, PC cells were cultivated in 96 wellplates at a density 2×10⁴ cells/well in DME/F12 medium supplemented withhuman fibronectin (2 μg/ml) and human transferrin (10 μg/ml). Increasingconcentrations of anti-GP88 IgG fraction were added to the wells afterthe cells were attached. Control cells were treated with equivalentconcentrations of non-immune IgG. Two days later, 0.25 mCi of³H-thymidine was added per well for 6 hrs. Cells were then harvested tocount ³H-thymidine incorporated into DNA as a measure for cellproliferation.

[0092] Moreover, when the expression of GP88 was specifically inhibitedby antisense GP88 cDNA in PC cells, the production of GP88 was reducedand these PC cells could no longer form tumors in syngeneic C3H mouse.In addition, these PC cells regained responsiveness to insulin. Withreference to FIG. 3 and Tables 1 and 2, C3H female mice were injectedsubcutaneously with 106 antisense GP88 transfected PC cells (asexplained below) or 10⁶ empty vector transfected PC cells. Mice weremonitored daily for tumor appearance. Photographs were taken 45 daysafter injection of the cells. The results show that mice injected withantisense GP88 PC cells do not develop tumors, in contrast to the miceinjected with empty vector transfected PC cells used as control. TABLE 1COMPARISON OF TUMORIGENIC PROPERTIES OF GP88 ANTISENSE TRANSFECTEDCELLS, CONTROL TRANSFECTED CELLS AND PC CELLS AVERAGE DAY OF NUMBER OFAVERAGE CELLS TUMOR MICE WITH TUMOR INJECTED DETECTION TUMORS WEIGHT (g)PC 15 ± 3.0 5/5 9.0 ± 3.2 P14 15 ± 3.7 5/5 7.8 ± 2.7 ASGP88 — 0/5 —

[0093] TABLE 2 COMPARISON OF PROPERTIES OF 1246, PC CELLS AND GP88ANTISENSE CELLS insulin GP88 antisense independence transfection 1246cells PC cells Antisense GP 88 cells insulin responsive forinsulin-independent for recovery of insulin growth and growthdifferentiation responsiveness for differentiation deficient growthautocrine production (differentiation?) of insulin-related factor cellsurface insulin cell surface insulin cell surface insulin receptorexpression receptor expression receptor expression high very lowelevated GP88 expression low GP88 expression GP88 expressionconstitutively high inhibited by antisense GP88 expression No inhibitionby serum inhibited by serum GP88 expression GP88 expression recovery ofinsulin regulated by insulin constitutive regulation for endogenous GP88expression non-tumorigenic highly tumorigenic non-tumorigenic

[0094] Comparison of the expression of GP88 indicates that in vivo GP88levels in tumors is dramatically higher than in normal tissues (FIG. 4).C3H mice were injected with 10⁶ PC cells. Tumor bearing mice wereeuthanized. Tumors, fat pads and connective tissue were collected. Celllysates were prepared by incubation in buffer containing detergent asdescribed above for FIG. 1. Protein concentration of tissue extracts wasdetermined, and equivalent amounts of proteins for each sample wereanalyzed by SDS-PAGE followed by Western blot analysis using anti-GP88antibody to measure the content of GP88 in tissue extracts. The resultsshowed that the level of GP88 in tumor extracts is at least 10-foldhigher than in surrounding connective and fat tissues.

[0095] In normal cells (1246 cells, fibroblasts), the expression of GP88is regulated, in particular by insulin, and inhibited by fetal bovineserum. In tumorigenic cells, a loss of regulation of normal growth leadsto the increased expression of GP88 and the acquisition of GP88dependence for growth. Therefore, inhibition of GP88 expression and/oraction is an effective approach to suppression of tumorigenesis.Detection of an elevated GP88 expression in biopsies provides diagnosticanalysis of tumors that are responsive to GP88 inhibition therapy.

[0096] GP88 is also a tumor-inducing factor in human cancers. As seen inthe 1246-3A cell line, a loss of responsiveness to insulin (or to IGF-I)and a concurrent increase in malignancy has been well documented inseveral human cancers including but not limited to breast cancers.Specifically, breast carcinoma is accompanied by the acquisition of aninsulin/IGF-I autocrine loop, which is also the starting point of thedevelopment of tumorigenic properties in the mouse model systemdiscussed above. Furthermore, GP88 expression is elevated in humanbreast carcinomas. More specifically, with reference to FIG. 5, humanGP88 was highly expressed in estrogen receptor positive and also inestrogen receptor negative insulin/IGF-I independent highly malignantcells. Also, GP88 is a potent growth factor for mammary epithelial cells(FIG. 6). The data in FIG. 5 was obtained by cultivating MCF7,MDA-MB-453 and MDA-MB-468 cells in DME/F12 medium supplemented with 10%fetal bovine serum (FBS). RNA was extracted from each cell line by theRNAzol method and poly-A⁺ RNA prepared. GP88 mRNA expression wasexamined by Northern blot analysis with 3 μg of poly-A⁺ RNA for eachcell line using a ³²P-labeled GP88 cDNA probe.

[0097] For Northern blot analysis of GP88 mRNA expression in rodentcells or tissues (mouse and rats), we used a mouse GP88 cDNA probe 311bp in length starting at nucleotide 551 to 862 (corresponding toamino-acid sequence 160 to 270). RNA can be extracted by a variety ofmethods (Sambrook, Molecular Biology manual: 35) well known to people ofordinary skill in the art. The method of choice was to extract RNA usingRNAzol (Cinnabiotech) or Trizol (Gibco-BRL) solutions which consists ofa single step extraction by guanidinium isothiocyanate andphenol-chloroform.

[0098] For Northern blot analysis of GP88 mRNA expression in human celllines, a 672 bp human GP88 cDNA probe was developed corresponding tonucleotide 1002 to 1674 (corresponding to amino-acid sequence 334-558)of human GP88. See example 8 for a detailed and specific description ofthe Northern blot analysis method used in the preferred embodiments.

[0099] With respect to FIG. 6, C57MG cells were cultivated in thepresence of increasing concentrations of GP88 purified from PC cellsconditioned medium (top panel), and recombinant GP88 expressed in insectcells (bottom panel), to demonstrate the growth stimulating effect ofincreasing concentrations of GP88 on the growth of the mouse mammaryepithelial cell line C57MG.

[0100] A correlation between FIG-I autocrine production and increasedmalignancy has also been well established for glioblastomas,teratocarcinomas and breast carcinomas. In these cancers, GP88expression is also elevated in human tumors when compared tonon-tumorigenic human fibroblasts and other human cell lines. GP88promotes the growth of mammary carcinoma cells.

Anti-GP88 Antibodies

[0101] The invention provides compositions for treating and diagnosingdiseases linked to increased expression of GP88. This also will apply totreatment and diagnosis of diseases linked to increased responsivenessto GP88. The compositions of this invention include anti-GP88 antibodieswhich neutralize the biological activity of GP88.

[0102] The present invention is also directed to an antibody specificfor an epitope of GP88 and the use of such antibody to detect thepresence or measure the quantity or concentration of GP88 molecule, afunctional derivative thereof or a homologue from different animalspecies in a cell, a cell or tissue extract, culture medium orbiological fluid. Moreover, anti-GP88 antibody can be used to targetcytotoxic molecules to a specific site.

[0103] For use as antigen for development of antibodies, the GP88protein naturally produced or expressed in recombinant form orfunctional derivative thereof, preferably having at least 9 amino-acids,is obtained and used to immunize an animal for production of polyclonalor monoclonal antibody. An antibody is said to be capable of binding amolecule if it is capable of reacting with the molecule to thereby bindthe molecule to the antibody. The specific reaction is meant to indicatethat the antigen will react in a highly selective manner with itscorresponding antibody and not with the multitude of other antibodieswhich may be evoked by other antigens.

[0104] The term antibody herein includes but is not limited to human andnon-human polyclonal antibodies, human and non-human monoclonalantibodies (mAbs), chimeric antibodies, anti-idiotypic antibodies(anti-IdAb) and humanized antibodies. Polyclonal antibodies areheterogeneous populations of antibody molecules derived either from seraof animals immunized with an antigen or from chicken eggs. Monoclonalantibodies (“mAbs”) are substantially homogeneous populations ofantibodies to specific antigens. mAbs may be obtained by methods knownto those skilled in the art (U.S. Pat. No. 4,376,110). Such antibodiesmay be of any immunological class including IgG, IgM, IgE, IgA, IgD andany subclass thereof. The hybridoma producing human and non-humanantibodies to GP88 may be cultivated in vitro or in vivo. For productionof a large amount of mAbs, in vivo is the presently preferred method ofproduction. Briefly, cells from the individual hybridomas are injectedintraperitoneally into pristane primed Balb/c mice or Nude mice toproduce ascites fluid containing high concentrations of the desiredmAbs. mAbs may be purified from such ascites fluids or from culturesupernatants using standard chromatography methods well known to thoseof skill in the art.

[0105] Human monoclonal Ab to human GP88 can be prepared by immunizingtransgenic mice expressing human immunoglobulin genes. Hybridomaproduced by using lymphocytes from these transgenic animals will producehuman immunoglobulin instead of mouse immunoglobulin.

[0106] Since most monoclonal antibodies are derived from murine sourceand other non-human sources, their clinical efficiency may be limiteddue to the immunogenicity of rodent mAbs administered to humans, weakrecruitment of effector function and rapid clearance from serum. Tocircumvent these problems, the antigen-binding properties of murineantibodies can be conferred to human antibodies through a process calledhumanization. A humanized antibody contains the amino-acid sequences forthe 6 complementarity-determining regions (CDRs) of the parent murinemAb which are grafted onto a human antibody framework. The low contentof non-human sequences in humanized antibodies (around 5%) has proveneffective in both reducing the immunogenicity and prolonging the serumhalf life in humans. Methods such as the ones using monovalent phagedisplay and combinatorial library strategy for humanization ofmonoclonal antibodies are now widely applied to the humanization of avariety of antibodies and are known to people skilled in the art. Thesehumanized antibodies and human antibodies developed with transgenicanimals as described above are of great therapeutic use for severaldiseases including but not limited to cancer.

[0107] Hybridoma supernatants and sera are screened for the presence ofantibody specific for GP88 by any number of immunoassays including dotblots and standard immunoassays (EIA or ELISA) which are well known inthe art. Once a supernatant has been identified as having an antibody ofinterest, it may be further screened by Western blotting to identify thesize of the antigen to which the antibody binds. One of ordinary skillin the art will know how to prepare and screen such hybridomas withoutundue experimentation in order to obtain a desired polyclonal or mAb.

[0108] Chimeric antibodies have different portions derived fromdifferent animal species. For example, a chimeric antibody might have avariable region from a murine mAab and a human immunoglobulin constantregion. Chimeric antibodies and methods for their production are alsoknown to those skilled in the art.

[0109] Accordingly, mAbs generated against GP88 may be used to inducehuman and non-human anti-IdAbs in suitable animals. Spleen cells fromsuch immunized mice are used to produce hybridomas secreting human ornon-human anti-Id mAbs. Further, the anti-Id mAbs can be coupled to acarrier such as Keyhole Limpet Hemocyanin (KLH) or bovine serum albumin(BSA) and used to immunize additional mice. Sera from these mice willcontain human or non-human anti-anti-IdAb that have the bindingproperties of the original mAb specific for a GP88 polypeptide epitope.The anti-Id mAbs thus have their own idiotypic epitopes or idiotypesstructurally similar to the epitope being evaluated.

[0110] The term antibody is also meant to include both intact moleculesas well as fragments thereof such as, for example, Fab and F(ab′)2,which are capable of binding to the antigen. Fab and F(ab′)2 fragmentslack the Fc fragment of intact antibody, clear more rapidly from thecirculation and may have less non-specific tissue binding than an intactantibody. Such fragments are typically produced by proteolytic cleavage,using enzymes such as papain (to generate Fab fragments) and pepsin (togenerate F(ab′)2 fragments). It will be appreciated that Fab and F(ab′)2and other fragments of the antibodies useful in the present inventionmay be used for the detection or quantitation of GP88, and for treatmentof pathological states related to GP88 expression, according to themethods disclosed herein for intact antibody molecules.

[0111] According to the present invention, antibodies that neutralizeGP88 activity in vitro can be used to neutralize GP88 activity in vivoto treat diseases associated with increased GP88 expression or increasedresponsiveness to GP88, such as but not limited to multiple myeloma. Asubject, preferably a human subject, suffering from multiple mycloma orother disease associated with increased GP88 expression is treated withan antibody to GP88. Such treatment may be performed in conjunction withother anti-cancer or anti-viral therapy. A typical regimen comprisesadministration of an effective amount of the antibody specific for GP88administered over a period of one or several weeks and including betweenabout one and six months. The antibody of the present invention may beadministered by any means that achieves its intended purpose. Forexample, administration may be by various routes including but notlimited to subcutaneous, intravenous, intradermal, intramuscular,intraperitoneal and oral. Parenteral administration can be by bolusinjection or by gradual perfusion over time. Preparations for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions and emulsions, which may contain auxiliary agents orexcipients known in the art. Pharmaceutical compositions such as tabletsand capsules can also be prepared according to routine methods. It isunderstood that the dosage of will be dependent upon the age, sex andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment and the nature of the effect desired. The ranges ofeffective doses provided below are not intended to limit the inventionand merely represent preferred dose ranges. However the most preferreddosage will be tailored to the individual subject as is understood anddeterminable by one skilled in the art. The total dose required for eachtreatment may be administered by multiple doses or in a single dose.Effective amounts of antibody are from about 0.01 μg to about 100 mg/kgbody weight and preferably from about 10 μg to about 50 mg/kg. Antibodymay be administered alone or in conjunction with other therapeuticsdirected to the same disease.

[0112] According to the present invention and concerning theneutralizing antibody, GP88 neutralizing antibodies can be used in alltherapeutic cases where it is necessary to inhibit GP88 biologicalactivity, even though there may not necessarily be a change in GP88expression, including cases where there is an overexpression of GP88cell surface receptors and this in turn results in an increasedbiological activity, or where there is an alteration in GP88 signalingpathways or receptors leading to the fact that the signaling pathwaysare always “turned on.” In one embodiment, the GP88 neutralizingantibodies are used to inhibit the growth of multiple myeloma cells.Neutralizing antibodies to growth factor and to growth factor receptorshave been successfully used to inhibit the growth of cells whoseproliferation is dependent on this growth factor. This has been the casefor IGF-I receptor in human breast carcinoma cells and bombesin for lungcancer. The antibody to GP88 can also be used to deliver compounds suchas, but not limited to, cytotoxic reagents such as toxins, oncotoxins,mitotoxins and immunotoxins, or antisense oligonucleotides, in order tospecifically target them to cells expressing or responsive to GP88.

[0113] One region that allows antigen to develop a neutralizing antibodyto GP88 is the 19 amino-acid region defined as K19T in the mouse GP88,and E19V in the human GP88 which is not located within theepithelin/granulin 6 kDa repeats but between these repeats, specificallybetween granulin A (epithelin 1) and granulin C in what is considered avariant region (see FIG. 10). Without wishing to be bound by theory, itis believed that the region important for the biological activity ofGP88 lies outside of the epithelin repeats.

[0114] The antibodies or fragments of antibodies useful in the presentinvention may also be used to quantitatively or qualitatively detect thepresence of cells which express the GP88 protein. This can beaccomplished by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below) with fluorescent microscopic, flowcytometric, or fluorometric detection. The reaction of antibodies andpolypeptides of the present invention may be detected by immunoassaymethods well known in the art.

[0115] The antibodies of the present invention may be employedhistologically as in light microscopy, immunofluorescence orimmunoelectron microscopy, for in situ detection of the GP88 protein intissues samples or biopsies. In situ detection may be accomplished byremoving a histological specimen from a patient and applying theappropriately labeled antibody of the present invention. The antibody(or fragment) is preferably provided by applying or overlaying thelabeled antibody (or fragment) to the biological sample. Through the useof such a procedure, it is possible to determine not only the presenceof the GP88 protein but also its distribution in the examined tissue.Using the present invention, those of ordinary skill in the art willreadily perceive that any wide variety of histological methods (such asstaining procedures) can be modified in order to achieve such in situdetection.

[0116] Assays for GP88 typically comprise incubating a biological samplesuch as a biological fluid, a tissue extract, freshly harvested orcultured cells or their culture medium in the presence of a detectablylabeled antibody capable of identifying the GP88 protein and detectingthe antibody by any of a number of techniques well known in the art.

[0117] The biological sample may be treated with a solid phase supportor carrier such as nitrocellulose or other solid support capable ofimmobilizing cells or cell particles or soluble proteins. The supportmay then be washed followed by treatment with the detectably labeledanti-GP88 antibody. This is followed by wash of the support to removeunbound antibody. The amount of bound label on said support may then bedetected by conventional means. By solid phase support is intended anysupport capable of binding antigen or antibodies such as but not limitedto glass, polystyrene polypropylene, nylon, modified cellulose, orpolyacrylamide.

[0118] The binding activity of a given lot of antibody to the GP88protein may be determined according to well known methods. Those skilledin the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

[0119] Detection of the GP88 protein or functional derivative thereofand of a specific antibody for the protein may be accomplished by avariety of immunoassays well known in the art such as enzyme linkedimmunoassays (EIA) or radioimmunoassays (RIA). Such assays are wellknown in the art and one of skill will readily know how to carry outsuch assays using the anti-GP88 antibodies and GP88 protein of thepresent invention.

[0120] Such immunoassays are useful to detect and quantitate GP88protein in serum or other biological fluid as well as in tissues, cells,cell extracts, or biopsies. In a preferred embodiment, the concentrationof GP88 is measured in a tissue specimen as a means for diagnosingcancer or other disease associated with increased expression of GP88.

[0121] The presence of certain types of cancers (e.g., multiple myeloma)and the degree of malignancy are said to be “proportional” to anincrease in the level of the GP88 protein. The term “proportional” asused herein is not intended to be limited to a linear or constantrelationship between the level of protein and the malignant propertiesof the cancer. The term “proportional” as used herein, is intended toindicate that an increased level of GP88 protein is related toappearance, recurrence or display of malignant properties of a cancer orother disease associated with increased expression of GP88 at ranges ofconcentration of the protein that can be readily determined by oneskilled in the art.

[0122] Another embodiment of the invention relates to evaluating theefficacy of anti-cancer or anti-viral drug or agent by measuring theability of the drug or agent to inhibit the expression or production ofGP88. The antibodies of the present invention are useful in a method forevaluating anti-cancer or anti-viral drugs in that they can be employedto determine the amount of the GP88 protein in one of theabove-mentioned immunoassays. Alternatively, the amount of the GP88protein produced is measured by bioassay (cell proliferation assay) asdescribed herein. The bioassay and immunoassay can be used incombination for a more precise assessment.

[0123] An additional embodiment is directed to an assay for diagnosingcancers or other diseases associated with an increase in GP88 expressionbased on measuring in a tissue or biological fluid the amount of mRNAsequences present that encode GP88 or a functional derivative thereof,preferably using an RNA-DNA hybridization assay. The presence of certaincancers and the degree of malignancy is proportional to the amount ofsuch mRNA present. For such assays the source of mRNA will be biopsiesand surrounding tissues. The preferred technique for measuring theamount of mRNA is a hybridization assay using DNA of complementaritybase sequence.

[0124] Another related embodiment is directed to an assay for diagnosingcancers or other diseases associated with an increase in GP88responsiveness based on measuring on a tissue biopsy whether treatmentwith anti-GP88 neutralizing antibody will inhibit its growth or otherbiological activity.

[0125] Another related embodiment is a method for measuring the efficacyof anticancer or anti-viral drug or agent which comprises the steps ofmeasuring the agent's effect on inhibiting the expression of mRNA forGP88. Similarly such method can be used to identify or evaluate theefficacy of GP88 antagonizing agents by measuring the ability of saidagent to inhibit the production of GP88 mRNA.

[0126] Nucleic acid detection assays, especially hybridization assays,can be based on any characteristic of the nucleic acid molecule such asits size, sequence, or susceptibility to digestion by restrictionendonucleases. The sensitivity of such assays can be increased byaltering the manner in which detection is reported or signaled to theobserver. A wide variety of labels have been extensively developed andused by those of ordinary skill in the art, including enzymatic,radioisotopic, fluorescent, chemical labels and modified bases.

[0127] One method for overcoming the sensitivity limitation of a nucleicacid for detection is to selectively amplify the nucleic acid prior toperforming the assay. This method has been referred as the “polymerasechain reaction” or PCR (U.S. Pat. Nos. 4,683,202 and 4,582,788). The PCRreaction provides a method for selectively increasing the concentrationof a particular nucleic acid sequence even when that sequence has notbeen previously purified and is present only in a single copy in aparticular sample.

GP88 Antisense Components

[0128] This invention also provides GP88 antisense components. Theconstitutive expression of antisense RNA in cells has been shown toinhibit the expression of more than 20 genes and the list continues togrow. Possible mechanisms for antisense effects are the blockage oftranslation or prevention of splicing, both of which have been observedin vitro. Interference with splicing allows the use of intron sequenceswhich should be less conserved and therefore result in greaterspecificity, inhibiting expression of a gene product of one species butnot its homologue in another species. Alternatively, nucleic acidsequences which inhibit or interfere with gene expression (e.g., RNAi,ribozymes, aptamers) can be used to inhibit or interfere with theactivity of RNA or DNA encoding GP88.

[0129] The term antisense component corresponds to an RNA sequence aswell as a DNA sequence coding therefor, which is sufficientlycomplementary to a particular mRNA molecule, for which the antisense RNAis specific, to cause molecular hybridization between the antisense RNAand the mRNA such that translation of the mRNA is inhibited. Suchhybridization can occur under in vivo conditions. The action of theantisense RNA results in specific inhibition of gene expression in thecells.

[0130] According to the present invention, transfection of B-cellleukemia cells with DNA antisense to the GP88 cDNA inhibits endogenousGP88 expression and inhibits tumorigenicity of the antisense cDNAtransfected cells. This antisense DNA must have sufficientcomplementarity, about 18-30 nucleotides in length, to the GP88 gene sothat the antisense RNA can hybridize to the GP88 gene (or mRNA) andinhibit GP88 gene expression regardless of whether the action is at thelevel of splicing, transcription, or translation. The degree ofinhibition is readily discernible to one skilled in the art withoutundue experimentation given the teachings herein and preferably issufficient to inhibit the growth of cells whose proliferation isdependent on the expression of GP88. One of ordinary skill in the artwill recognize that the antisense RNA approach is but a number of knownmechanisms which can be employed to block specific gene expression.

[0131] The antisense components of the present invention may behybridizable to any of several portions of the target GP88 cDNA,including the coding sequence, 3′ or 5′ untranslated regions, or otherintronic sequences, or to GP88 mRNA. As is readily discernible by one ofordinary skill in the art, the minimal amount of homology required bythe present invention is that sufficient to result in hybridization tothe GP88 DNA or mRNA and in inhibition of transcription of the DNA, ortranslation or function of the mRNA, preferably without affecting thefunction of other mRNA molecules and the expression of other unrelatedgenes.

[0132] Antisense RNA is delivered to a cell by transformation ortransfection via a vector, including retroviral vectors and plasmids,into which has been placed DNA encoding the antisense RNA with theappropriate regulatory sequences including a promoter to result inexpression of the antisense RNA in a host cell. Stable transfection ofvarious antisense expression vectors containing GP88 cDNA fragments inthe antisense orientation have been performed. One can also deliverantisense components to cells using a retroviral vector. Delivery canalso be achieved by liposomes.

[0133] For purpose of antisense technology for in vivo therapy, thecurrently preferred method is to use antisense oligonucleotides, insteadof performing stable transfection of an antisense cDNA fragmentconstructed into an expression vector. Antisense oligonucleotides havinga size of 15-30 bases in length and with sequences hybridizable to anyof several portions of the target GP88 cDNA, including the codingsequence, 3′ or 5′ untranslated regions, or other intronic sequences, orto GP88 mRNA, are preferred. Sequences for the antisenseoligonucleotides to GP88 are preferably selected as being the ones thathave the most potent antisense effects. Factors that govern a targetsite for the antisense oligonucleotide sequence are related to thelength of the oligonucleotide, binding affinity, and accessibility ofthe target sequence. Sequences may be screened in vitro for potency oftheir antisense activity by measuring inhibition of GP88 proteintranslation and GP88 related phenotype, e.g., inhibition of cellproliferation in cells in culture. In general it is known that mostregions of the RNA (5′ and 3′ untranslated regions, AUG initiation,coding, splice junctions and introns) can be targeted using antisenseoligonucleotides.

[0134] The preferred GP88 antisense oligonucleotides are thoseoligonucleotides which are stable, have a high resilience to nucleases(enzymes that could potentially degrade oligonucleotides), possesssuitable pharmacokinetics to allow them to traffic to disease tissue atnon-toxic doses, and have the ability to cross through plasma membranes.

[0135] Phosphorothioate antisense oligonucleotides may be used.Modifications of the phosphodiester linkage as well as of theheterocycle or the sugar may provide an increase in efficiency. Withrespect to modification of the phosphodiester linkage, phophorothioatemay be used. An N3′-P5′ phosphoramidate linkage has been described asstabilizing oligonucleotides to nucleases and increasing the binding toRNA. Peptide nucleic acid (PNA) linkage is a complete replacement of theribose and phosphodiester backbone and is stable to nucleases, increasesthe binding affinity to RNA, and does not allow cleavage by RNAse H. Itsbasic structure is also amenable to modifications that may allow itsoptimization as an antisense component. With respect to modifications ofthe heterocycle, certain heterocycle modifications have proven toaugment antisense effects without interfering with RNAse H activity. Anexample of such modification is C-5 thiazole modification. Finally,modification of the sugar may also be considered. 2′-O-propyl and2′-methoxyethoxy ribose modifications stabilize oligonucleotides tonucleases in cell culture and in vivo. Cell culture and in vivo tumorexperiments using these types of oligonucleotides targeted to c-raf-1resulted in enhanced potency.

[0136] The delivery route will be the one that provides the bestantisense effect as measured according to the criteria described above.In vitro cell culture assays and in vivo tumor growth assays usingantisense oligonucleotides have shown that delivery mediated by cationicliposomes, by retroviral vectors and direct delivery are efficient.Another possible delivery mode is targeting using antibody to cellsurface markers for the tumor cells. Antibody to GP88 or to its receptormay serve this purpose.

Inhibiting the Growth of Hematopoietic Malignant Cells

[0137] Preferred embodiments of the invention are directed to methodsand compositions for reducing, interfering with, and/or inhibiting thegrowth and proliferation of hematopoietic malignant cells. Hematopoicticcells are divided into three categories: erythroid, myeloid and lymphoidcells. The erythroid cells are red blood cells and their precursors.Myeloid cells include monocytes, granulocytes, basophils, cosinophilsand megakaryocytes. Myeloma is a type of cancer originating from myeloidcells (monocytes). Hematopoietic malignant cells include, but are notlimited to leukemias (e.g., ALL (Acute lymphoblastic leukemia), AML(acute myelogenous leukemia), CML (chronic myelogenous leukemia), acutebilineage leukemia, acute undifferentiated leukemia, chronic lymphocyticleukemia, juvenile chronic myelogenous leukemia, prolymphocyticleukemia, MDS (myelodysplastic syndromes), acquired idiopathicsideroblastic anemia, acute myelofibrosis, chronic myelomonocyticleukemia, essential thrombocythemia, myelodysplastic disorders,myelofibrosis mycloid metaplasia, paroxysmal nocturnal hemoglobinuria,polycythemia vera, refractory anemia, refractory anemia with excessblasts (RAEB), refractory anemia with excess blasts in transformation(RAEB-T)), and lymphomas (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma,plasma cell dyscrasia, multiple mycloma, plasma cell leukemia,waldenstrom macroglobulinemia).

[0138] As described above, hematopoietic malignant cells expresselevated levels of GP88. The present invention demonstrates that GP88 isthe first growth factor shown to be a prognostic indicator ofhematopoietic malignancies (e.g., B-cell leukemias such as multiplemyeloma), and that GP88 antagonists reduce, inhibit, and/or interferewith the growth of hematopoietic malignant cells.

[0139] As shown in FIGS. 16 and 17, GP88 protein (FIG. 16) and mRNA(FIG. 17) is overexpressed in human multiple myeloma cell lines ARP-1and RPMI 8226 and human B cell lines Raji and Daudi and not expressed inhuman T-cell lines Jurkat and KOPTI-K1. In addition, GP88 stimulatesgrowth and increases the percent survival of multiple myeloma cells. Thelive cell density (i.e., growth) and viability of RPMI 8226 multiplemyeloma cells increased in a dose dependant manner in response toincreased amounts of GP88 (FIGS. 18A and 18B). As shown in FIG. 18A, thelive cell density of RPMI 8226 cells increased by 3-fold in the presenceof 200 ng/ml of GP88. Likewise, the percent survival of RPMI 8226 cellsincreased by 2-fold after 48 hours in the presence of 100 or 200 ng/mlof GP88. The growth and viability response of RPMI 8226 mycloma cells toGP88 is similar to that of myeloma cells to IL-6 (compare FIG. 18A col.3 to col. 4 and FIG. 18B col. 3 to col. 4). Similar results wereobtained with ARP-1 multiple myeloma cells. The live cell density ofARP-1 cells more than doubled in the presence of 200 ng/ml of GP88.(FIG. 19A). The percent survival of ARP-1 multiple myeloma cells doubledafter 48 hours in the presence of 200 ng/ml of GP88. IL-6 also doubledboth the live cell density and percent survival after 48 hours of ARP-1cells. (FIGS. 19A and 19B). Reducing, inhibiting, or interfering withthe growth stimulatory and survival effects of GP88 on myeloma cellsreduces the growth and survival of multiple myeloma cells, providing atherapeutic benefit to multiple myeloma patients.

[0140] In one embodiment of the invention, a method of inhibiting,reducing, or interfering with the growth of hematopoietic malignantcells (e.g., B-cell leukemias cells such as mycloma cells) by contactinghematopoietic malignant cells with a GP88 antagonist is provided. Asdescribed above, GP88 antagonists (e.g., anti-GP88 antibodies) inhibitthe growth of myeloma cells. In another embodiment of the invention, thehematopoietic malignant cells are human myeloma cells.

[0141] GP88 antagonists (e.g., anti-GP88 antibodies or antibodyfragments, and GP88 small molecules) bind to GP88 secreted from the celland inhibit and/or interfere with the biological activity of GP88. GP88antagonists can, for example, bind to GP88 and prevent GP88 from bindingto its receptor on the cell surface. GP88 antagonists (e.g., anti-GP88antisense polynucleotides) can also enter the cell and inhibit orinterfere with the expression of the GP88 protein. For example,anti-GP88 antisense polynucleotides can hybridize with mRNA encodingGP88 and block translation of the GP88 protein. Alternatively, the GP88antagonist may be conjugated or linked to another molecule capable ofinterfering or inhibiting cell growth (e.g., toxins, antibodies,antibody fragments, and nucleic acids). GP88 antagonists also caninterfere with the biological activity of GP88 by binding to a moleculeother than GP88. For example, GP88 antagonists can bind to, inhibit,and/or interfere with the activity of the GP88 receptor and thusinterfere with the binding of GP88 to its receptor.

[0142] The term “GP88 antagonist” refers to any composition thatinhibits or blocks GP88 expression, production or secretion, or anycomposition that inhibits or blocks the biological activity of GP88including, but not limited to, anti-GP88 antibodies, anti-GP88 antisensepolynucleotides, anti-GP88 receptor antibodies, anti-GP88 smallmolecules. In one embodiment of the invention, the GP88 antagonist is ananti-GP88 antibody or antibody fragment. The term “antibody fragment”refers to any section, portion, or part of an antibody that retains theantigen binding properties of the antibody. Anti-GP88 antibodies alsoinclude antibody fragments, humanized antibodies, humanized antibodyfragments and can be made as described above.

[0143] The term “contacting” refers to delivering GP88 antagonist tohematopoietic malignant cells (e.g., leukemia cells of B-cell lineage)wherein the GP88 antagonist can interact with the cell either directly(e.g., binding to GP88 inside the cell) or indirectly (e.g., binding toGP88 and preventing GP88 from directly contacting myeloma cells). A GP88antagonist may be injected into the blood stream of a patient sufferingfrom a hematopoietic malignancy to bind GP88 and prevent GP88 fromstimulating hematopoietic malignant cell growth. GP88 antagonist mayalso be microinjected into a cell by shooting pellets coated with GP88antagonist inside the cell in order to prevent secretion of GP88.Hematopoietic malignant cells may also be transfected with nucleic acidencoding a GP88 antagonist. Alternatively, patients can be treated witha GP88 small molecule antagonist to block GP88 activity. Contactinghematopoietic malignant cells with GP88 antagonist blocks the activityof GP88 and therefore inhibits, reduces, and/or interferes with thegrowth of the cells. GP88 antagonists such as anti-GP88 antibodies andanti-GP88 antisense nucleic acids can be made and administered by anysuitable mechanism (e.g., injection, and aerosol) as described above.

[0144] Administration of GP88 antagonists to hematopoietic malignantcells significantly reduces the growth of the cells. For example,anti-GP88 neutralizing antibody inhibits the growth of RPMI 8226multiple mycloma cells by about 50% while treatment of the same cellswith non-immuno rabbit IgG did not show any significant inhibition ofcell growth. (FIG. 20). Addition of exogenous GP88 reversed theinhibitory effect of the anti-GP88 neutralizing antibody. (FIG. 20). Thereversal of GP88 antagonist induced growth inhibition by the addition ofexogenous GP88 demonstrates that GP88 is a growth factor for myelomacells. The growth of myeloma cells can be measured by several methodsincluding, but not limited to, measuring the live cell density in vitroby staining cells with trypan blue, uptake of radioactive nucleotides,cell mass, BudR incorporation, ELISA, cell metabolism, spectroscopy, anddirect measurement of the dimensions of a tumor mass.

[0145] Preferred embodiments of the invention are also directed tomethods of diagnosing B-cell leukemia by detecting GP88 in tissuesamples containing B-cells (e.g., blood, bone marrow, lymph, spleen,liver). The presence of GP88 in tissue samples containing B-cellsindicates B-cell leukemia. GP88 protein or nucleic acid can be detectedas described above. Also provided are methods of diagnosing B-cellleukemia by detecting the presence of GP88 in B-cells. The presence ofGP88 in B-cells indicates B-cell leukemia.

[0146] In another embodiment of the invention, the presence of GP88 inbone marrow cells indicates the presence of multiple myeloma cells. Thepresence of immunoglobulin lambda or kappa light chains in bone marrowcells is a marker for neoplastic or potentially neoplastic myelomacells. Hitzman et al., Immunoperoxidase staining of bone marrowsections, Cancer 48(11):2438-46 (1981). Immunostaining bone marrowsections for the presence of lambda or kappa immunoglobulin light chainsallows for detection of myeloma cases that are difficult to diagnosesuch as nonsecretory myeloma. Id. As shown in Table 1, such myelomacells that stain positive for kappa or lambda light chains also stainpositive for GP88. TABLE 1 Expression of Ig light chain and GP88 in bonemarrow smears From multiple myeloma patients Patients Ig κ chain Ig λchain GP88  1 + − +  2 − − −  3 − + +  4 + − +  5a − − −  5b + − +  6 −− −  7 − + +  8 + − +  9 − − − 10 + − + 11 + − + 12 + − +

[0147] GP88 is not detected in bone marrow cells from patients inremission or in cells that do not express kappa or lambda immunoglobulinlight chains. Furthermore, patients in remission for multiple mycloma(e.g., Patient 5a) do not express GP88. Patient 5a relapsed anddisplayed the symptoms of multiple myeloma (Patient 5b). Patient 5b waspositive for both the kappa light chain and GP88. Thus, GP88 serves as abiological marker for multiple myeloma. An example of a triple stain forthe presence of a control (DAPI), kappa/lambda light chains, and GP88 inthe same patient sample is shown in FIGS. 25A, 25B, and 25Crespectively. Detecting the presence of GP88 in bone marrow cells isindicative of whether multiple myeloma cells are present. The presenceof GP88 can be detected by GP88 antagonists (e.g., anti-GP88 antibodies,anti-GP88 nucleic acid) using a variety of methodologies including, butnot limited to, immunostaining, immunofluorescence, in situhybridization, western blot, northern blot, and southern blot.

The Presence of GP88 Indicates Whether a Patient is Responding orResponsive to Anti-Cancer Therapy

[0148] Anti-cancer agents such as glucocorticoids and glucocorticoidanalogs (e.g., dexamethasone, prednisolone, methylprednisolone,hydrocortisone, betamethasone, prednisone, fludrocortisone, cortisone,corticosterone, triamcinolone, and paramethasone) alone or incombination with chemotherapy (e.g., alkylating agents) are used totreat patients with hematopoictic malignancies (e.g., B-cell leukemia).However, certain patients may not be responsive to anti-cancer therapy.In addition, it is well known that patients that are initiallyresponsive to anti-cancer therapy develop resistance and no longerrespond to the drugs.

[0149] For example, prolonged systemic exposure to glucocorticoids mayhave severe adverse side effects such as: (1) endocrine and metabolicdisturbances including, but not limited to, Cushing-like syndrome,hirsutism, menstrual irregularities, premature epiphyseal closure,secondary adrenocortical and pituitary unresponsiveness, decreasedglucose tolerance, and negative nitrogen and calcium balance; (2) fluidand electrolyte disturbances such as sodium and fluid retention,hypertension, potassium loss, and hypokalaemic alkalosis; (3)musculo-skeletal effects (e.g., myopathy, abdominal distension,osteoporosis, aseptic necrosis of femoral and humeral heads); (4)gastrointestinal effects including gastric and duodenal ulceration,perforation, and hemorrhage; (5) dermatological effects such as impairedwound healing, skin atrophy, striae, petechiae and ecchymoses, bruising,facial erythema, increased sweating, and acne; (6) central nervoussystem effects (e.g., psychic disturbances ranging from euphoria tofrank psychotic manifestations, convulsions, pseudotumor cerebri (benignintracranial hypertension) with vomiting and papilloedema); (7)ophthalmic effects including glaucoma, increased intraocular pressure,posterior subcapsular cataracts; and (8) immunosuppressive effects suchas increased susceptibility to infections, decreased responsiveness tovaccination and skin tests. Thus, unnecessary exposure to anti-cancertherapy (e.g., glucocorticoids, such as dexamethasone), should belimited to the extent possible to avoid causing complications anddiscomfort without significant positive benefits.

[0150] Dexamethasone induces apoptosis of multiple myeloma cells. Asshown in FIG. 26, dexamethasone also inhibits GP88 protein expression.GP88 protein expression was measured by Western blot analysis ofconditioned media collected by ARP-1 cell cultures in the presence andabsence of dexamethasone alone and in combination with IL-6 (FIG. 26).Dexamethasone significantly inhibited the expression of GP88 protein.The addition of exogenous GP88 overcomes the apoptosis-inducing effectsof dexamethasone (FIGS. 27A and 27B). As shown in FIGS. 27A and 27B,GP88 significantly increased both cell growth (FIG. 27A) and cellviability (FIG. 27B) of ARP-1 cells treated with dexamethasone. FIG. 28shows that GP88 significantly reduces the cleavage of an apoptosismarker PARP (Poly (ADP-ribose) polymerase) in dexamethasone-treatedARP-1 cells. Cleavage of PARP into two fragments is a marker of cellapoptosis. Thus, GP88 has an anti-apoptotic effect and can inhibitdexamethasone-induced killing of B-cell leukemia cells.

[0151] Increased levels of GP88 in MM cells are responsible for thetransition of MM cells to a glucocorticoid resistant form. As shown inFIG. 29, cells transfected with GP88 (ARP-1/PCDGF) produced ten timesmore GP88 than untransfected cells or control ARP-1 cells that weretransfected with empty vector (ARP-1/EV). (FIG. 29). MM cellstransfected with GP88 show an increased growth rate and viability(resistance to the killing effect of dexamethasone) (FIGS. 30A and 30B).As shown in FIG. 30A, the ARP-1/PCDGF cells had a higher growth rate andwere more resistant to the apoptotic effects of dexamethasone (columns 1and 2) than the ARP-1/empty vector control cells (columns 3 and 4).Likewise, the ARP-1/PCDGF cells showed increased viability in responseto the addition of dexamethasone (columns 1 and 2) that the ARP-1/emptyvector cells. Thus, the presence of GP88 indicates that B-cells are orhave become dexamethasone-resistant.

[0152] Methods of determining whether a patient is responding orresponsive to anti-cancer therapy by detecting the presence of GP88 in atissue sample containing B-cells are also provided by the invention. Theterm “responding” to anti-cancer therapy refers to patients who arereceiving anti-cancer therapy. One embodiment of the invention willdetermine if such patients should continue to receive anti-cancertherapy. The term “responsive” to anti-cancer therapy refers to patientswho are not yet receiving anti-cancer therapy. Another embodiment of theinvention will determine if such patients should begin to receiveanti-cancer therapy. Increased levels of GP88 in tissue samples (e.g.,detectable increase in the level of GP88) containing B-cells over timeindicate that the patient is not responding or responsive to anti-cancertherapy (e.g., glucocorticoids such as dexamethasone). Alternatively,increased levels of GP88 in B-cells compared to normal or peripheraltissues is sufficient to indicate that the patient is not responding orresponsive to glucocorticoid therapy. GP88 protein and/or nucleic acids(e.g., DNA or RNA encoding GP88) can be detected as described above(e.g., using anti-GP88 antibodies, antisense nucleic acids). In anotherembodiment, the GP88 level in an individual patient's B cells or tissuescontaining B-cells can be periodically monitored. An increased level ofGP88 in a patient's B-cells or in tissues containing B-cells over timeindicates that the patient is not responding or responsive toanti-cancer therapy.

Recombinant GP88

[0153] The present invention is also directed to DNA expression systemsfor expressing a recombinant GP88 polypeptide or a functional derivativethereof substantially free of other mammalian DNA sequences. Such DNAmay be double or single stranded. The DNA sequence should preferablyhave about 20 or more nucleotides to allow hybridization to anotherpolynucleotide. In order to achieve higher specificity of hybridization,characterized by the absence of hybridization to sequences other thanthose encoding the GP88 protein or a homologue or functional derivativethereof, a length of at least 50 nucleotides is preferred.

[0154] The present invention is also directed to the above DNAmolecules, expressible vehicles or vectors as well as hosts transfectedor transformed with the vehicles and capable of expressing thepolypeptide. Such hosts may be prokaryotic, preferably bacteria, oreukaryotic, preferably yeast, mammalian or insect cells. A preferredvector system includes baculovirus expressed in insect cells. The DNAcan be incorporated into host organisms by transformation, transduction,transfection, infection or related processes known in the art. Inaddition to DNA and mRNA sequences encoding the GP88 polypeptide, theinvention also provides methods for expression of the nucleic acidsequence. Further, the genetic sequences and oligonucleotides allowidentification and cloning of additional polypeptides having sequencehomology to the polypeptide GP88 described here.

[0155] An expression vector is a vector which (due to the presence ofappropriate transcriptional and/or translational control sequences) iscapable of expressing a DNA (or cDNA) molecule which has been clonedinto the vector and thereby produces a polypeptide or protein.Expression of the cloned sequence occurs when the expression vector isintroduced into an appropriate host cell. If a prokaryotic expressionvector is employed, then the appropriate host cell would be anyprokaryotic cell capable of expressing the cloned sequence. Similarly,if an eukaryotic expression system is employed, then the appropriatehost cell would be any eukaryotic cell capable of expressing the clonedsequence. Baculovirus vector, for example, can be used to clone GP88cDNA and subsequently express the cDNA in insect cells.

[0156] A DNA sequence encoding GP88 polypeptide or its functionalderivatives may be recombined with vector DNA in accordance withconventional techniques including blunt-ended or staggered ended terminifor ligation, restriction enzyme digestion to provide appropriatetermini, filling in cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and ligation with proper enzymeligases. Techniques for such manipulations are discussed in (35).

[0157] A nucleic acid molecule is capable of expressing a polypeptide ifit contains nucleotide sequences which contain transcriptional andtranslational regulatory information and such sequences are operablylinked to nucleotide sequences which encode the polypeptide. An operablelinkage is a linkage in which the regulatory DNA sequences and the DNAsequence sought to be expressed are connected in such a way as to permitgene expression. The precise nature of the regulatory regions needed forgene expression may vary from organism to organism but shall in generalinclude a promoter region, which in prokaryotes contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which when transcribed into RNA will signal theinitiation of protein synthesis. Such regions will normally includethose 5′ non-coding sequences involved with the initiation oftranscription, translation such as the TATA box, capping sequence, CAATsequence and the like.

[0158] If desired, the 3′ non-coding region to the gene sequenceencoding the protein may be obtained by described methods (screeningappropriate cDNA library or PCR amplification). This region may beretained for the presence of transcriptional termination regulatorysequences such as termination and polyadenylation. Thus, by retainingthe 3′ region naturally contiguous to the DNA sequence coding for theprotein, the transcriptional termination signals may be provided. Wherethe transcription termination signals are not provided or satisfactorilyfunctional in the expression host cells, then a 3′ region from anothergene may be substituted.

[0159] Two DNA sequences such as a promoter region sequence and GP88encoding sequence are said to be operably linked if the nature of thelinkage between the sequences does not result in the introduction of aframe-shift mutation or interfere with the ability of the promotersequence to direct transcription of the polypeptide gene sequence.

[0160] The promoter sequences may be prokaryotic, eukaryotic or viral.Suitable promoters are inducible, repressible or constitutive. Examplesof suitable prokaryotic promoters are reviewed by.

[0161] Eukaryotic promoters include but are not limited to the promoterfor the mouse methallothionein I gene, the TK promoter of Herpes Virus,the gene gal4 promoter, the SV40 early promoter, the mouse mammary tumorvirus (MMTV) promoter, and the cytomegalovirus (CMV) promoter. Strongpromoters are preferred. Examples of such promoters are those whichrecognize the T3, SP6 and T7 polymerases, the PL promoter ofbacteriophage lambda, the recA promoter, the promoter of the mousemethallothionein I gene, the SV40 promoter and the CMV promoter.

[0162] It is to be understood that application of the teachings of thepresent invention to a specific problem or environment will be withinthe capability of one having ordinary skill in the art in light of theteachings contained herein. The present invention is more fullyillustrated by the following non-limiting examples.

EXAMPLE 1 Cell Lines and Reagents

[0163] Daudi, Raji, KOPM-28, ARP-1, RPMI 8226, Jurkat, KOPT-K1, andHL-60 were obtained from the American Type Culture Collection (ATCC,Manhassas, Va.). RPMI 1640 medium, FBS, and Trizol was obtained fromInvitrogen life technologies (Carlsbad, Calif.). Alexa 456 conjugatedgoat anti mouse IgG F(ab′)2 and Alexa 488 conjugated goat anti rabbitIgG F(ab′)2 were obtained from Molecular Probes (Eugene, Oreg.). IL-6was obtained from Upstate Biotechnology Inc. (Lack Placid, N.Y.).PD98059, anti phosph-MAPK antibody, anti phosph-Akt antibody, anti Aktantibody, anti phosph-tyr-STAT3 were obtained from New England Biolabs(Beverly, Mass.). Anti STAT3 was obtained from BD Biosciences. Anti MAPKantibody was obtained from Santa Cruz Biotechnology (Santa Cruze,Calif.). LY194002 was obtained from Biomol (Plymouth Meeting, Pa.).Supersignal Western chemiluminescent substrate was obtained from Pierce(Rockford, Ill.). Immobilon-P transfer membranes were obtained fromMillipore (Bedford, Mass.). Monoclonal antibodies to anti human κ or λlight chains were obtained from Dako (Carpinteria, Calif.). Protein Asepharose was obtained from Amersham Pharmacia Biotech (Piscataway,N.J.). GP88 and anti-GP88 antibody were purified in our lab and aredescribed in U.S. Pat. No. ______ [insert]. All other reagents wereobtained from Sigma.

GP88 Protein Expression

[0164] Daudi, Raji, KOPM-28, ARP-1, RPMI 8226, Jurkat, HL-60, andKOPT-K1 were cultured at a density of 1×10⁵ cells/ml in RPMI mediumsupplemented with 10% FBS. Until the cells reach a density of 1×10⁶cells/ml, the culture media equivalent to 1.5×10⁷ live cells werecollected to measure GP88 protein expression. Immunoprecipitaion andWestern Blot analysis were carried out as described previously (18)using 50 ug/ml anti-GP88 F(ab′) conjugated to HRP as the detectingantibody.

Northern Blot Analysis

[0165] RPMI 8226 and ARP-1 cell were cultured in 10% FBS RPMI medium.RNA isolation was carried out using Trizol. Northern Blot analysis wascarried out as described previously (18).

Cell Growth and Survival Assay

[0166] RPMI 8226 or ARP-1 cells were cultured in 10% FBS RPMI. Beforethe assay, cells were washed by serum free RPMI 1640 twice and culturedin serum free RPMI 1640 medium for 24 hours. GP88 or IL-6 was added tomedia at indicated concentration. Live cell density and viability weredetermined by trypan blue exclusion and cell counting. Experiments werecarried out in triplicate sets with results expressed as mean±SD.

Anti-GP88 Neutralizing Assay

[0167] RPMI 8226 cells were cultured in 10% FBS RPMI 1640, washed byRPMI 1640 twice, and cultured in RPMI 1640 media at 1×10⁵ cells/ml.Affinity purified anti-GP88, non-immuno rabbit IgG, or affinity purifiedanti-GP88 antibody with GP88 was added as appropriate. After 48 hours,live cell density was checked by trypan blue staining and cell counting.Experiments were carried out in triplicate sets and the result wasexpressed as mean±SD.

MAPK Assay

[0168] ARP-1 cells were cultured in 10% FBS RPMI 1640 medium, washed byRPMI 1640 twice, and resuspended at 2.5×10⁵ live cells/ml in RPMI 1640.After overnight starvation, ARP-1 cells were either treated with orwithout 30 μM PD98059 for 60 min. GP88 was added to final concentrationof 200 ng/ml except wells for negative controls. Ten milliliters of cellculture was used for each sample. After ten minutes of incubation, thecells were lysed by loading buffer. Cell lysates were separated on a12.5% SDS-PAGE gel. The phosph-MAPK and total MAPK proteins weredetected by anti-phoph-MAPK and anti MAPK antibodies respectively usingWestern blot analysis.

Akt Assay

[0169] ARP-1 cells were cultured in 10% FBS RPMI 1640 medium, washed inRPMI 1640 twice, and resuspended at 2.5×10⁵ live cells/ml in RPMI 1640.After overnight starvation, ARP-1 cells were either treated with orwithout 50 μM LY194002 for ten minutes. GP88 was added to theexperimental wells on a microtiter plate at a final concentration of 200ng/ml. GP88 was not added to control wells. Ten milliliters of cellculture was used for each sample. After ten minutes of incubation, cellswere lysed by loading buffer. Cell lysates were separated on a 12.5%SDS-PAGE gel. The phosph-Akt and total Akt proteins were detected byanti-phoph-Akt and anti Akt antibodies respectively using Western blotanalysis.

STAT3 Assay

[0170] ARP-1 cells were cultured in 10% FBS RPMI 1640 medium, washedtwice in RPMI 1640 medium, and resuspended at 2.5×10⁵ live cells/ml inRPMI 1640. After starvation of the cell culture overnight, ARP-1 cellswere treated with 200 ng/ml GP88 or 10 ng/ml IL-6 for 15 min. Cells werelysed by loading buffer and separated on a 7.5% SDS-PAGE. 3×10⁶ cellswere used for each sample. The phosph-tyr-STAT3 and total STAT3 proteinswere detected by anti-phoph-tyr-STAT3 and anti STAT3 antibodiesrespectively using Western blot analysis.

Immunocytochemistry Studies

[0171] Bone marrow smears obtained from multiple mycloma patients at theUniversity of Maryland Greenbaum Cancer Center were fixed for 15 minuteson ice with 2% paraformaldehyde in PBS, washed by PBS, and permeabilizedwith 0.2% Triton X100 for 15 minutes at room temperature. The slideswere stained with 0.85 μg/ml rabbit anti-human GP88 antibody at roomtemperature for 1 hour, washed by PBS, and incubated with secondary 2μg/ml Alexa 488-conjugated goat anti rabbit IgG F(ab′)2 at roomtemperature for 1 hour. These slides were also stained with 0.25 μg/mlmonoclonal antibodies to anti human K or X light chains at roomtemperature for 1 hour, washed by PBS, and followed by incubation with 1μg/ml Alexa4S6 conjugated goat anti mouse IgG F(ab′)2 at roomtemperature for 1 hour. Finally, samples were stained by 0.5 μg/ml DAPIat room temperature for 15 minutes. Stained bone marrow samples wereobserved with Olympus BX40 fluorescence microscope equipped with 100Wmercury lamp and appropriate filters.

GP88 Expression in Human Hematological Cell Line

[0172] We examined GP88 expression in several human leukemic cell lines.Samples examined were standardized to the same cell number. FIG. 16shows GP88 protein expression was high in human B cell lines (Raji andDaudi) and human MM cell lines (ARP-1 and RPMI 8226). In contrast, noGP88 was produced in human T cell lines (Jurkat and kOPT-K1) andpromyelocytic leukemia (HL-60). A low level of GP88 was found inmacrophage cell line (KOPM-28). HL-60 is a promyclocytic cell line thatcan be induced to differentiate terminally to granulocyte-like cells ormonocyte/macrophage-like cells upon exposure to different reagents (19).These results show that GP88 is preferentially expressed byhematological malignancies of B cell lineage. The level of GP88 mRNAexpression in the MM cell lines ARP-1 and RPMI 8226 is shown in FIG. 17.

GP88 Function in Two Human MM Cell Lines: RPMI 8226 and ARP-1

[0173] The effect of exogenously added GP88 on the growth and survivalof RPMI 8226 (FIG. 18) and ARP-1 (FIG. 19) was examined and compared toIL-6, a known paracrine growth stimulator of MM cell growth. As shown inFIG. 3A, RPMI 8226 cells were starved in RPMI medium only for 24 hours,then GP88 or IL-6 was added to medium. After 24 hour treatment 50 ng/ml(5.7×10−7 M), 100 ng/ml (1.1×10−6 M), 200 ng/ml (2.3×10−6 M) GP88, and10 ng/ml (4.5×10−7 M) IL-6 stimulated the growth of RPMI 8226 cells by1.3, 1.5, 1.5, and 1.6-fold, respectively. After 48 hour treatment, 50,100, 200 ng/ml GP88, and 10 ng/ml IL-6 stimulated the growth of RPMI8226 cells by 1.7, 2.5, 2.6, and 2.8 fold, respectively. These data showthat GP88 stimulates the growth of RPMI 8226 cells in a dose and timedependent fashion similarly to IL-6. In addition to stimulating thegrowth of human MM cells, exogenous GP88 also stimulated cell survivalof RPMI 8226 similarly to IL-6 (FIG. 18B).

[0174] Dex-sensitive ARP-1 cells exhibited similar growth and survivaleffects in response to GP88. As shown in FIG. 19A, ARP-1 cells werestarved in serum-free medium for 24 hours, then GP88 or IL-6 was addedto medium. At 24 hours of treatment, 200 ng/ml GP88 and 10 ng/ml IL-6stimulated the growth of ARP-1 cells by 1.3 and 1.4 fold, respectively.After 48 hours of treatment, 200 ng/ml GP88 and 10 ng/ml IL-6 stimulatedthe growth of ARP-1 cells by 2.3 and 2.6 fold, respectively. Similarlyexogenous GP88 also stimulated cell survival of ARP-1 (FIG. 19B).

Effect of Anti-GP88 Neutralizing Antibody on the Growth of RPMI 8226Cells

[0175] In order to check whether GP88 produced and secreted by MM cellswas required for cell growth, we examined the effect of anti-GP88neutralizing antibody on the growth of RPMI 8226 cells. We have shownpreviously that this antibody was able to inhibit the proliferation ofbreast cancer cells overexpressing GP88 (20). As shown in FIG. 20,treatment of RPMI 8226 cell with 200 μg/ml affinity purified anti-GP88antibody inhibited RPMI 8226 cell growth by about 50% in serum freecondition. However, treatment of RPMI 8226 cells with 200 μg/mlnon-immuno rabbit IgG did not significantly inhibit RPMI 8226 cellgrowth. Addition of exogenous 200 ng/ml GP88 prevented the inhibitioneffect of anti-GP88 antibody. These results show that GP88 stimulated MMcell growth in an autocrine fashion.

Signaling Pathway Stimulated by GP88 in ARP-1 Cells

[0176] We examined signal pathways involved in growth factor signaltransduction to determine their role, if any, in the GP88 signaltransduction pathway in MM cells. MAPK signal pathway plays a key rolein proliferation process and MAPK activity is stimulated in response tomany different growth factors (21, 22). PI3K signal pathway is primarilyassociated with survival and cell growth regulation (23, 24). FIG. 21shows that stimulation of ARP-1 cell growth and survival by 200 ng/ml ofGP88 was blocked by the MEK inhibitor PD98059 at 30 μM. FIG. 22 shows200 ng/ml GP88 activated the phosphorylation of Erk1 and Erk2 and thisphosphorylation was also inhibited by 30 μM PD98059. Together, theseresults show that GP88 activated MAPK signal pathway in ARP-1 cells andthat MAPK was responsible for stimulation of cell growth by GP88. Todetermine the role of the PI3K signal pathway, we assessed thephosphorylation of Akt. Akt contains an amino-terminal pleckstrinhomology (PH) domain that binds phosphorylated lipids at the membrane inresponse to activation of PI3 kinase (25, 26). FIG. 23 shows that GP88stimulates the phosphorylation of Akt in ARP-1 cells and thisphosphorylation was inhibited by PI3 K inhibitor LY294002 at 50 μM.These results showed that GP88 activated PI3 kinase signal pathway inARP-1 cells.

[0177] MAPK and JAK/STAT pathways are two important signaling pathwaysin human MM cells induced by IL-6 (6). In order to check whether GP88activates JAK/STAT pathways in human MM cells, the phosphorylation ofSTAT3 was assessed following stimulation of MM cells by 200 ng/ml GP88or 10 ng/ml IL-6. As shown in FIG. 24, only IL-6, but not GP88,stimulates the phosphorylation of STAT3. These data suggest that GP88does not activate the JAK/STAT3 pathway in human MM cells.

Immunocytochemistry Studies of Human Patient Bone Marrow Smears

[0178] We examined GP88 expression in 13 bone marrow biopsy samples frompatients with multiple myeloma by immunocytochemistry staining of GP88and human κ/λ light chains. The presence κ or λ light chains in bonemarrow cells is a marker of myeloma cells (27). Table 1 demonstratesthat GP88 was overexpressed in bone marrow smears of MM patients.Staining of the samples with anti-human κ or λ light chains showed thatthe myeloma cells that stained positive for GP88 were positive for κ orλ light chains indicating that the cells that overexpress GP88 in bonemarrow smears of MM patients are the multiple myeloma cells.GP88-positive cells were not observed in the bone marrow smears frompatients in remission (patients 2, 5a, 6 and 9) where κ or λ lightchain-positive cells were not detected. It is important to note thatwhen the relapse of MM disease occurred in patient 5a, GP88 expressionwas detected in the bone marrow samples and co-localized with cellsexpressing K light chains (5b in, Table 1). The κ/λ chain positive cellsshowed 100% GP88 positive staining by counting 100 κ/λ chain positivecells. A typical triple staining by DAPI, κ/λ chain, and GP88 is shownin FIG. 25. These data clearly indicated that GP88 expression isassociated with myeloma cells from all MM patients examined andcorrelated well with the presence of the disease.

The Effects of Dexamethasone (Dex) and IL-6 Effect on GP88 ProteinExpression in ARP-1 Cells

[0179] ARP-1 cells were seeded in 10% CT-FBS RPMI 1640 in the presenceof 10−7M Dex or 10 ng/ml IL-6 added alone or in combination. Controlcells were cultivated with vehicle medium that did not contain Dex orIL-6. After 48 hours, the cell culture medium was changed to RPMI 1640for 24 hours and the conditioned medium was collected. The GP88 secretedin the conditioned medium was measured by immunoprecipitation andwestern blot analysis (FIG. 26). Dex inhibited the expression of GP88 inDex-treated ARP-1 cells.

The Effects of Exogenous Addition of GP88 and IL-6 on Dex-Induced CellDeath

[0180] ARP-1 cells were cultured in media containing 10% CT-FBS RPMI1640 medium in the presence of 10-7 M Dex, 200 ng/ml GP88, or 10 ng/mlIL-6 added alone or in combination. After 48 hours, the live celldensity and cell viability were checked by trypan blue exclusion andcounting with a hemocytometer. FIG. 27A shows the effect on live celldensity and FIG. 27B shows the effect on cell viability. GP88 increasedthe growth and viability of Dex-treated ARP-1 cells.

The Effects of GP88 on PARP Cleavage in ARP-1 Cells

[0181] ARP-1 cells were seeded and treated with 10-7 M Dex, 200 ng/mlPCDGF, and 10 ng/ml IL-6 as described above. Cells were collected at 24hours and 48 hours. ARP-1 cells were lysed and 100 μg protein per lanewere used to analyze PARP cleavage by SDS-PAGE and western blotanalysis. GP88 inhibited the apoptotic effects of Dex on Dex-treatedARP-1 cells (FIG. 28).

Overexpression of GP88 in ARP-1 Cells

[0182] Dexamethasone-sensitive human MM cell line ARP-1 was transfectedwith expression vector pcDNA3 containing a CMV promoter, a neomycinresistant gene, and GP88 cDNA by electroporation. Transfected cells wereselected in the presence of G418. GP88 expression in the cell culturemedia was detected by immunoprecipitation and western blot analysis. Asshown in FIG. 29, ARP-1 cells transfected with GP88 (ARP-1/PCDGF) hadelevated levels of GP88 compared to cells transfected with an emptyexpression vector (ARP-1/EV).

The Effects of Dex on ARP-1 and GP88 Overexpressed ARP-1 cells

[0183] ARP-1/PCDGF cells that overexpress GP88 and ARP-1/EV werecultured in 10% CT-FBS RPMI with or without 10−7 M Dex. Cell density (A)and viability (B) were measured after 24 hours. As shown in FIGS. 30Aand 30B, Dex-induced reduction in cell growth and viability wassignificantly reduced in cells with elevated levels of GP88 (columns 1and 2) compared to cells that did not express GP88 (columns 3 and 4).

The Effects of GP88 Over Expression Dex-Induced PARP Cleavage

[0184] Empty vector control and PCDGF overexpressing ARP-1 cells weretreated with or without 10-7 M Dex. After 48 hours, the cells were lysedto measure the expression of intact and cleaved PARP (FIG. 31). Cellsoverexpressing GP88 (ARP-1/PCDGF) showed greatly reduced cleavage ofPARP compared to cells that did not express GP88. Thus, GP88 inhibitsthe apoptotic effects of dexamethasone on ARP-1 cells.

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[0218]

1 17 1 2137 DNA Mouse epithelin/granulin CDS (23)..(1789) The sequenceis identical to that of the published mouse granulin except for onenucleotide (T instead of G) at position 1071 of GP88 cDNA (position 1056of mouse granulin). 1 cggaccccga cgcagacaga cc atg tgg gtc ctg atg agctgg ctg gcc ttc 52 Met Trp Val Leu Met Ser Trp Leu Ala Phe 1 5 10 gcggca ggg ctg gta gcc gga aca cag tgt cca gat ggg cag ttc tgc 100 Ala AlaGly Leu Val Ala Gly Thr Gln Cys Pro Asp Gly Gln Phe Cys 15 20 25 cct gttgcc tgc tgc ctt gac cag gga gga gcc aac tac agc tgc tgt 148 Pro Val AlaCys Cys Leu Asp Gln Gly Gly Ala Asn Tyr Ser Cys Cys 30 35 40 aac cct cttctg gac aca tgg cct aga ata acg agc cat cat cta gat 196 Asn Pro Leu LeuAsp Thr Trp Pro Arg Ile Thr Ser His His Leu Asp 45 50 55 ggc tcc tgc cagacc cat ggc cac tgt cct gct ggc tat tct tgt ctt 244 Gly Ser Cys Gln ThrHis Gly His Cys Pro Ala Gly Tyr Ser Cys Leu 60 65 70 ctc act gtg tct gggact tcc agc tgc tgc ccg ttc tct aag ggt gtg 292 Leu Thr Val Ser Gly ThrSer Ser Cys Cys Pro Phe Ser Lys Gly Val 75 80 85 90 tct tgt ggt gat ggctac cac tgc tgc ccc cag ggc ttc cac tgt agt 340 Ser Cys Gly Asp Gly TyrHis Cys Cys Pro Gln Gly Phe His Cys Ser 95 100 105 gca gat ggg aaa tcctgc ttc cag atg tca gat aac ccc ttg ggt gct 388 Ala Asp Gly Lys Ser CysPhe Gln Met Ser Asp Asn Pro Leu Gly Ala 110 115 120 gtc cag tgt cct gggagc cag ttt gaa tgt cct gac tct gcc acc tgc 436 Val Gln Cys Pro Gly SerGln Phe Glu Cys Pro Asp Ser Ala Thr Cys 125 130 135 tgc att atg gtt gatggt tcg tgg gga tgt tgt ccc atg ccc cag gcc 484 Cys Ile Met Val Asp GlySer Trp Gly Cys Cys Pro Met Pro Gln Ala 140 145 150 tct tgc tgt gaa gacaga gtg cat tgc tgt ccc cat ggg gcc tcc tgt 532 Ser Cys Cys Glu Asp ArgVal His Cys Cys Pro His Gly Ala Ser Cys 155 160 165 170 gac ctg gtt cacaca cga tgc gtt tca ccc acg ggc acc cac acc cta 580 Asp Leu Val His ThrArg Cys Val Ser Pro Thr Gly Thr His Thr Leu 175 180 185 cta aag aag ttccct gca caa aag acc aac agc gca gtg tct ttg cct 628 Leu Lys Lys Phe ProAla Gln Lys Thr Asn Ser Ala Val Ser Leu Pro 190 195 200 ttt tct gtc gtgtgc cct gat gct aag acc cag tgt ccc gat gat tct 676 Phe Ser Val Val CysPro Asp Ala Lys Thr Gln Cys Pro Asp Asp Ser 205 210 215 acc tgc tgt gagcta ccc act ggg aag tat ggc tgc tgt cca atg ccc 724 Thr Cys Cys Glu LeuPro Thr Gly Lys Tyr Gly Cys Cys Pro Met Pro 220 225 230 aat gcc atc tgctgt tcc gac cac ctg cac tgc tgc ccc cag gac act 772 Asn Ala Ile Cys CysSer Asp His Leu His Cys Cys Pro Gln Asp Thr 235 240 245 250 gta tgt gacctg atc cag agt aag tgc cta tcc aag aac tac acc acg 820 Val Cys Asp LeuIle Gln Ser Lys Cys Leu Ser Lys Asn Tyr Thr Thr 255 260 265 gat ctc ctgacc aag ctg cct gga tac cca gtg aag gag gtg aag tgc 868 Asp Leu Leu ThrLys Leu Pro Gly Tyr Pro Val Lys Glu Val Lys Cys 270 275 280 gac atg gaggtg agc tgc cct gaa gga tat acc tgc tgc cgc ctc aac 916 Asp Met Glu ValSer Cys Pro Glu Gly Tyr Thr Cys Cys Arg Leu Asn 285 290 295 act ggg gcctgg ggc tgc tgt cca ttt gcc aag gcc gtg tgt tgt gac 964 Thr Gly Ala TrpGly Cys Cys Pro Phe Ala Lys Ala Val Cys Cys Asp 300 305 310 gat cac attcat tgc tgc ccg gca ggg ttt cag tgt cac aca gag aaa 1012 Asp His Ile HisCys Cys Pro Ala Gly Phe Gln Cys His Thr Glu Lys 315 320 325 330 gga acctgc gaa atg ggt atc ctc caa gta ggg tgg atg aag aag gtc 1060 Gly Thr CysGlu Met Gly Ile Leu Gln Val Gly Trp Met Lys Lys Val 335 340 345 ata gccccc ctc cgc ctg cca gac cca cag atc ttg aag agt gat aca 1108 Ile Ala ProLeu Arg Leu Pro Asp Pro Gln Ile Leu Lys Ser Asp Thr 350 355 360 cct tgtgat gac ttc act agg tgt cct aca aac aat acc tgc tgc aaa 1156 Pro Cys AspAsp Phe Thr Arg Cys Pro Thr Asn Asn Thr Cys Cys Lys 365 370 375 ctc aattct ggg gac tgg ggc tgc tgt ccc atc cca gag gct gtc tgc 1204 Leu Asn SerGly Asp Trp Gly Cys Cys Pro Ile Pro Glu Ala Val Cys 380 385 390 tgc tcagac aac cag cat tgc tgc cct cag ggc ttc aca tgt ctg gct 1252 Cys Ser AspAsn Gln His Cys Cys Pro Gln Gly Phe Thr Cys Leu Ala 395 400 405 410 cagggg tac tgt cag aag gga gac aca atg gtg gct ggc ctg gag aag 1300 Gln GlyTyr Cys Gln Lys Gly Asp Thr Met Val Ala Gly Leu Glu Lys 415 420 425 atacct gcc cgc cag aca acc ccg ctc caa att gga gat atc ggt tgt 1348 Ile ProAla Arg Gln Thr Thr Pro Leu Gln Ile Gly Asp Ile Gly Cys 430 435 440 gaccag cat acc agc tgc cca gta ggg caa acc tgc tgc cca agc ctc 1396 Asp GlnHis Thr Ser Cys Pro Val Gly Gln Thr Cys Cys Pro Ser Leu 445 450 455 aaggga agt tgg gcc tgc tgc cag ctg ccc cat gct gtg tgc tgt gag 1444 Lys GlySer Trp Ala Cys Cys Gln Leu Pro His Ala Val Cys Cys Glu 460 465 470 gaccgg cag cac tgt tgc ccg gcc ggg tac acc tgc aac gtg aag gcg 1492 Asp ArgGln His Cys Cys Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala 475 480 485 490agg acc tgt gag aag gat gtc gat ttt atc cag cct ccc gtg ctc ctg 1540 ArgThr Cys Glu Lys Asp Val Asp Phe Ile Gln Pro Pro Val Leu Leu 495 500 505acc ctc ggc cct aag gtt ggg aat gtg gag tgt gga gaa ggg cat ttc 1588 ThrLeu Gly Pro Lys Val Gly Asn Val Glu Cys Gly Glu Gly His Phe 510 515 520tgc cat gat aac cag acc tgt tgt aaa gac agt gca gga gtc tgg gcc 1636 CysHis Asp Asn Gln Thr Cys Cys Lys Asp Ser Ala Gly Val Trp Ala 525 530 535tgc tgt ccc tac cta aag ggt gtc tgc tgt aga gat gga cgt cac tgt 1684 CysCys Pro Tyr Leu Lys Gly Val Cys Cys Arg Asp Gly Arg His Cys 540 545 550tgc ccc ggt ggc ttc cac tgt tca gcc agg gga acc aag tgt ttg cga 1732 CysPro Gly Gly Phe His Cys Ser Ala Arg Gly Thr Lys Cys Leu Arg 555 560 565570 aag aag att cct cgc tgg gac atg ttt ttg agg gat ccg gtc cca aga 1780Lys Lys Ile Pro Arg Trp Asp Met Phe Leu Arg Asp Pro Val Pro Arg 575 580585 ccg cta ctg taaggaaggg ctacagactt aaggaactcc acagtcctgg 1829 Pro LeuLeu gaaccctgtt ccgagggtac ccactactca ggcctcccta gcgcctcctc ccctaacgtc1889 tccccggcct actcatcctg agtcacccta tcaccatggg aggtggagcc tcaaactaaa1949 accttctttt atggaaagaa ggctctggcc aaaagccccg tatcaaactg ccatttcttc2009 cggtttctgt ggaccttgtg gccaggtgct cttcccgagc cacaggtgtt ctgtgagctt2069 gcttgtgtgt gtgtgcgcgt gtgcgtgtgt tgctccaata aagtttgtac gctttctgaa2129 aaaaaaaa 2137 2 589 PRT Mouse epithelin/granulin 2 Met Trp Val LeuMet Ser Trp Leu Ala Phe Ala Ala Gly Leu Val Ala 1 5 10 15 Gly Thr GlnCys Pro Asp Gly Gln Phe Cys Pro Val Ala Cys Cys Leu 20 25 30 Asp Gln GlyGly Ala Asn Tyr Ser Cys Cys Asn Pro Leu Leu Asp Thr 35 40 45 Trp Pro ArgIle Thr Ser His His Leu Asp Gly Ser Cys Gln Thr His 50 55 60 Gly His CysPro Ala Gly Tyr Ser Cys Leu Leu Thr Val Ser Gly Thr 65 70 75 80 Ser SerCys Cys Pro Phe Ser Lys Gly Val Ser Cys Gly Asp Gly Tyr 85 90 95 His CysCys Pro Gln Gly Phe His Cys Ser Ala Asp Gly Lys Ser Cys 100 105 110 PheGln Met Ser Asp Asn Pro Leu Gly Ala Val Gln Cys Pro Gly Ser 115 120 125Gln Phe Glu Cys Pro Asp Ser Ala Thr Cys Cys Ile Met Val Asp Gly 130 135140 Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Ser Cys Cys Glu Asp Arg 145150 155 160 Val His Cys Cys Pro His Gly Ala Ser Cys Asp Leu Val His ThrArg 165 170 175 Cys Val Ser Pro Thr Gly Thr His Thr Leu Leu Lys Lys PhePro Ala 180 185 190 Gln Lys Thr Asn Ser Ala Val Ser Leu Pro Phe Ser ValVal Cys Pro 195 200 205 Asp Ala Lys Thr Gln Cys Pro Asp Asp Ser Thr CysCys Glu Leu Pro 210 215 220 Thr Gly Lys Tyr Gly Cys Cys Pro Met Pro AsnAla Ile Cys Cys Ser 225 230 235 240 Asp His Leu His Cys Cys Pro Gln AspThr Val Cys Asp Leu Ile Gln 245 250 255 Ser Lys Cys Leu Ser Lys Asn TyrThr Thr Asp Leu Leu Thr Lys Leu 260 265 270 Pro Gly Tyr Pro Val Lys GluVal Lys Cys Asp Met Glu Val Ser Cys 275 280 285 Pro Glu Gly Tyr Thr CysCys Arg Leu Asn Thr Gly Ala Trp Gly Cys 290 295 300 Cys Pro Phe Ala LysAla Val Cys Cys Asp Asp His Ile His Cys Cys 305 310 315 320 Pro Ala GlyPhe Gln Cys His Thr Glu Lys Gly Thr Cys Glu Met Gly 325 330 335 Ile LeuGln Val Gly Trp Met Lys Lys Val Ile Ala Pro Leu Arg Leu 340 345 350 ProAsp Pro Gln Ile Leu Lys Ser Asp Thr Pro Cys Asp Asp Phe Thr 355 360 365Arg Cys Pro Thr Asn Asn Thr Cys Cys Lys Leu Asn Ser Gly Asp Trp 370 375380 Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cys Ser Asp Asn Gln His 385390 395 400 Cys Cys Pro Gln Gly Phe Thr Cys Leu Ala Gln Gly Tyr Cys GlnLys 405 410 415 Gly Asp Thr Met Val Ala Gly Leu Glu Lys Ile Pro Ala ArgGln Thr 420 425 430 Thr Pro Leu Gln Ile Gly Asp Ile Gly Cys Asp Gln HisThr Ser Cys 435 440 445 Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Lys GlySer Trp Ala Cys 450 455 460 Cys Gln Leu Pro His Ala Val Cys Cys Glu AspArg Gln His Cys Cys 465 470 475 480 Pro Ala Gly Tyr Thr Cys Asn Val LysAla Arg Thr Cys Glu Lys Asp 485 490 495 Val Asp Phe Ile Gln Pro Pro ValLeu Leu Thr Leu Gly Pro Lys Val 500 505 510 Gly Asn Val Glu Cys Gly GluGly His Phe Cys His Asp Asn Gln Thr 515 520 525 Cys Cys Lys Asp Ser AlaGly Val Trp Ala Cys Cys Pro Tyr Leu Lys 530 535 540 Gly Val Cys Cys ArgAsp Gly Arg His Cys Cys Pro Gly Gly Phe His 545 550 555 560 Cys Ser AlaArg Gly Thr Lys Cys Leu Arg Lys Lys Ile Pro Arg Trp 565 570 575 Asp MetPhe Leu Arg Asp Pro Val Pro Arg Pro Leu Leu 580 585 3 19 PRT mousegranulin PEPTIDE (1)..(19) Internal peptide of mouse GP88 used to raisethe antisera against the GP88 used in the immunoaffinity step. 3 Lys LysVal Ile Ala Pro Arg Arg Leu Pro Asp Pro Gln Ile Leu Lys 1 5 10 15 SerAsp Thr 4 12 PRT mouse granulin PEPTIDE (1)..(12) Internal peptide ofmouse GP88 used to raise the antisera against the GP88 used in theimmunoaffinity step. 4 Pro Asp Ala Lys Thr Gln Cys Pro Asp Asp Ser Thr 15 10 5 14 PRT mouse granulin PEPTIDE (1)..(14) Internal peptide of mouseGP88 used to raise the antisera against the GP88 used in theimmunoaffinity step. 5 Ser Ala Arg Gly Thr Lys Cys Leu Arg Lys Lys IlePro Arg 1 5 10 6 19 PRT Human granulin PEPTIDE (1)..(19) Internalpeptide of human GP88 used to develop neutralizing anti-human GP88monoclonal antibody. 6 Glu Lys Ala Pro Ala His Leu Ser Leu Pro Asp ProGln Ala Leu Lys 1 5 10 15 Arg Asp Val 7 14 PRT Human granulin PEPTIDE(1)..(14) Internal peptide of human GP88 used to develop neutralizinganti-human GP88 monoclonal antibody. 7 Ala Arg Arg Gly Thr Lys Cys LeuArg Arg Glu Ala Pro Arg 1 5 10 8 24 DNA mammalian primer (1)..(24)Internal peptide of CMV promoter used as PCR primer. 8 cctacttggcagtacatcta cgta 24 9 27 DNA mammalian primer (1)..(27) GP88 cDNA startcodon used as oligonucleotide PCR primer. 9 cgagaattca ggcagaccatgtgggtc 27 10 27 DNA mammalian primer (1)..(27) Antisense primeroligonucleotide primer 10 cgagaattca ggcagaccat gtgggtc 27 11 23 DNAmammalian primer (1)..(23) Antisense primer oligonucleotide primer 11ctgacggttc actaaacgag ctc 23 12 25 DNA mammalian primer (1)..(25) primer12 ggatccacgg agttgttacc tgatc 25 13 25 DNA mammalian primer (1)..(25)oligonucleotide PCR primer 13 gaattcgcag gcagaccatg tggac 25 14 21 DNAmammalian primer (1)..(21) Antisense oligonucleotide to human GP88 14gggtccacat ggtctgcctg c 21 15 24 DNA mammalian primer (1)..(24)Antisense oligonucleotide to human GP88 15 gccaccagcc ctgctgttaa ggcc 2416 2095 DNA Human GP88 cDNA CDS (13)..(1791) Nucleotide sequence ofhuman granulin/epithelin precursor (human GP88). Human Granulin GenebankM75161. 16 cgcaggcaga cc atg tgg acc ctg gtg agc tgg gtg gcc tta aca gcaggg 51 Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly 1 5 10 ctggtg gct gga acg cgg tgc cca gat ggt cag ttc tgc cct gtg gcc 99 Leu ValAla Gly Thr Arg Cys Pro Asp Gly Gln Phe Cys Pro Val Ala 15 20 25 tgc tgcctg gac ccc gga gga gcc agc tac agc tgc tgc cgt ccc ctt 147 Cys Cys LeuAsp Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg Pro Leu 30 35 40 45 ctg gacaaa tgg ccc aca aca ctg agc agg cat ctg ggt ggc ccc tgc 195 Leu Asp LysTrp Pro Thr Thr Leu Ser Arg His Leu Gly Gly Pro Cys 50 55 60 cag gtt gatgcc cac tgc tct gcc ggc cac tcc tgc atc ttt acc gtc 243 Gln Val Asp AlaHis Cys Ser Ala Gly His Ser Cys Ile Phe Thr Val 65 70 75 tca ggg act tccagt tgc tgc ccc ttc cca gag gcc gtg gca tgc ggg 291 Ser Gly Thr Ser SerCys Cys Pro Phe Pro Glu Ala Val Ala Cys Gly 80 85 90 gat ggc cat cac tgctgc cca cgg ggc ttc cac tgc agt gca gac ggg 339 Asp Gly His His Cys CysPro Arg Gly Phe His Cys Ser Ala Asp Gly 95 100 105 cga tcc tgc ttc caaaga tca ggt aac aac tcc gtg ggt gcc atc cag 387 Arg Ser Cys Phe Gln ArgSer Gly Asn Asn Ser Val Gly Ala Ile Gln 110 115 120 125 tgc cct gat agtcag ttc gaa tgc ccg gac ttc tcc acg tgc tgt gtt 435 Cys Pro Asp Ser GlnPhe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val 130 135 140 atg gtc gat ggctcc tgg ggg tgc tgc ccc atg ccc cag gct tcc tgc 483 Met Val Asp Gly SerTrp Gly Cys Cys Pro Met Pro Gln Ala Ser Cys 145 150 155 tgt gaa gac agggtg cac tgc tgt ccg cac ggt gcc ttc tgc gac ctg 531 Cys Glu Asp Arg ValHis Cys Cys Pro His Gly Ala Phe Cys Asp Leu 160 165 170 gtt cac acc cgctgc atc aca ccc acg ggc acc cac ccc ctg gca aag 579 Val His Thr Arg CysIle Thr Pro Thr Gly Thr His Pro Leu Ala Lys 175 180 185 aag ctc cct gcccag agg act aac agg gca gtg gcc ttg tcc agc tcg 627 Lys Leu Pro Ala GlnArg Thr Asn Arg Ala Val Ala Leu Ser Ser Ser 190 195 200 205 gtc atg tgtccg gac gca cgg tcc cgg tgc cct gat ggt tct acc tgc 675 Val Met Cys ProAsp Ala Arg Ser Arg Cys Pro Asp Gly Ser Thr Cys 210 215 220 tgt gag ctgccc agt ggg aag tat ggc tgc tgc cca atg ccc aac gcc 723 Cys Glu Leu ProSer Gly Lys Tyr Gly Cys Cys Pro Met Pro Asn Ala 225 230 235 acc tgc tgctcc gat cac ctg cac tgc tgc ccc caa gac act gtg tgt 771 Thr Cys Cys SerAsp His Leu His Cys Cys Pro Gln Asp Thr Val Cys 240 245 250 gac ctg atccag agt aag tgc ctc tcc aag gag aac gct acc acg gac 819 Asp Leu Ile GlnSer Lys Cys Leu Ser Lys Glu Asn Ala Thr Thr Asp 255 260 265 ctc ctc actaag ctg cct gcg cac aca gtg ggc gat gtg aaa tgt gac 867 Leu Leu Thr LysLeu Pro Ala His Thr Val Gly Asp Val Lys Cys Asp 270 275 280 285 atg gaggtg agc tgc cca gat ggc tat acc tgc tgc cgt cta cag tcg 915 Met Glu ValSer Cys Pro Asp Gly Tyr Thr Cys Cys Arg Leu Gln Ser 290 295 300 ggg gcctgg ggc tgc tgc cct ttt acc cag gct gtg tgc tgt gag gac 963 Gly Ala TrpGly Cys Cys Pro Phe Thr Gln Ala Val Cys Cys Glu Asp 305 310 315 cac atacac tgc tgt ccc gcg ggg ttt acg tgt gac acg cag aag ggt 1011 His Ile HisCys Cys Pro Ala Gly Phe Thr Cys Asp Thr Gln Lys Gly 320 325 330 acc tgtgaa cag ggg ccc cac cag gtg ccc tgg atg gag aag gcc cca 1059 Thr Cys GluGln Gly Pro His Gln Val Pro Trp Met Glu Lys Ala Pro 335 340 345 gct cacctc agc ctg cca gac cca caa gcc ttg aag aga gat gtc ccc 1107 Ala His LeuSer Leu Pro Asp Pro Gln Ala Leu Lys Arg Asp Val Pro 350 355 360 365 tgtgat aat gtc agc agc tgt ccc tcc tcc gat acc tgc tgc caa ctc 1155 Cys AspAsn Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cys Gln Leu 370 375 380 acgtct ggg gag tgg ggc tgc tgt cca atc cca gag gct gtc tgc tgc 1203 Thr SerGly Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cys 385 390 395 tcggac cac cag cac tgc tgc ccc cag cga tac acg tgt gta gct gag 1251 Ser AspHis Gln His Cys Cys Pro Gln Arg Tyr Thr Cys Val Ala Glu 400 405 410 gggcag tgt cag cga gga agc gag atc gtg gct gga ctg gag aag atg 1299 Gly GlnCys Gln Arg Gly Ser Glu Ile Val Ala Gly Leu Glu Lys Met 415 420 425 cctgcc cgc cgc ggt tcc tta tcc cac ccc aga gac atc ggc tgt gac 1347 Pro AlaArg Arg Gly Ser Leu Ser His Pro Arg Asp Ile Gly Cys Asp 430 435 440 445cag cac acc agc tgc ccg gtg ggc gga acc tgc tgc ccg agc cag ggt 1395 GlnHis Thr Ser Cys Pro Val Gly Gly Thr Cys Cys Pro Ser Gln Gly 450 455 460ggg agc tgg gcc tgc tgc cag ttg ccc cat gct gtg tgc tgc gag gat 1443 GlySer Trp Ala Cys Cys Gln Leu Pro His Ala Val Cys Cys Glu Asp 465 470 475cgc cag cac tgc tgc ccg gct ggc tac acc tgc aac gtg aag gct cga 1491 ArgGln His Cys Cys Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Arg 480 485 490tcc tgc gag aag gaa gtg gtc tct gcc cag cct gcc acc ttc ctg gcc 1539 SerCys Glu Lys Glu Val Val Ser Ala Gln Pro Ala Thr Phe Leu Ala 495 500 505cgt agc cct cac gtg ggt gtg aag gac gtg gag tgt ggg gaa gga cac 1587 ArgSer Pro His Val Gly Val Lys Asp Val Glu Cys Gly Glu Gly His 510 515 520525 ttc tgc cat gat aac cag acc tgc tgc cga gac aac cga cag ggc tgg 1635Phe Cys His Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln Gly Trp 530 535540 gcc tgc tgt ccc tac gcc cag ggc gtc tgt tgt gct gat cgg cgc cac 1683Ala Cys Cys Pro Tyr Ala Gln Gly Val Cys Cys Ala Asp Arg Arg His 545 550555 tgc tgt cct gct ggc ttc cgc tgc gca cgc agg ggt acc aag tgt ttg 1731Cys Cys Pro Ala Gly Phe Arg Cys Ala Arg Arg Gly Thr Lys Cys Leu 560 565570 cgc agg gag gcc ccg cgc tgg gac gcc cct ttg agg gac cca gcc ttg 1779Arg Arg Glu Ala Pro Arg Trp Asp Ala Pro Leu Arg Asp Pro Ala Leu 575 580585 aga cag ctg ctg tgagggacag tactgaagac tctgcagccc tcgggacccc 1831 ArgGln Leu Leu 590 actcggaggg tgccctctgc tcaggcctcc ctagcacctc cccctaaccaaattctccct 1891 ggaccccatt ctgagctccc catcaccatg ggaggtgggg cctcaatctaaggcccttcc 1951 ctgtcagaag ggggttgagg caaaagccca ttacaagctg ccatcccctccccgtttcag 2011 tggaccctgt ggccaggtgc ttttccctat ccacaggggt gtttgtgtgttgggtgtgct 2071 ttcaataaag tttgtcactt tctt 2095 17 593 PRT Human GP88cDNA 17 Met Trp Thr Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala1 5 10 15 Gly Thr Arg Cys Pro Asp Gly Gln Phe Cys Pro Val Ala Cys CysLeu 20 25 30 Asp Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg Pro Leu Leu AspLys 35 40 45 Trp Pro Thr Thr Leu Ser Arg His Leu Gly Gly Pro Cys Gln ValAsp 50 55 60 Ala His Cys Ser Ala Gly His Ser Cys Ile Phe Thr Val Ser GlyThr 65 70 75 80 Ser Ser Cys Cys Pro Phe Pro Glu Ala Val Ala Cys Gly AspGly His 85 90 95 His Cys Cys Pro Arg Gly Phe His Cys Ser Ala Asp Gly ArgSer Cys 100 105 110 Phe Gln Arg Ser Gly Asn Asn Ser Val Gly Ala Ile GlnCys Pro Asp 115 120 125 Ser Gln Phe Glu Cys Pro Asp Phe Ser Thr Cys CysVal Met Val Asp 130 135 140 Gly Ser Trp Gly Cys Cys Pro Met Pro Gln AlaSer Cys Cys Glu Asp 145 150 155 160 Arg Val His Cys Cys Pro His Gly AlaPhe Cys Asp Leu Val His Thr 165 170 175 Arg Cys Ile Thr Pro Thr Gly ThrHis Pro Leu Ala Lys Lys Leu Pro 180 185 190 Ala Gln Arg Thr Asn Arg AlaVal Ala Leu Ser Ser Ser Val Met Cys 195 200 205 Pro Asp Ala Arg Ser ArgCys Pro Asp Gly Ser Thr Cys Cys Glu Leu 210 215 220 Pro Ser Gly Lys TyrGly Cys Cys Pro Met Pro Asn Ala Thr Cys Cys 225 230 235 240 Ser Asp HisLeu His Cys Cys Pro Gln Asp Thr Val Cys Asp Leu Ile 245 250 255 Gln SerLys Cys Leu Ser Lys Glu Asn Ala Thr Thr Asp Leu Leu Thr 260 265 270 LysLeu Pro Ala His Thr Val Gly Asp Val Lys Cys Asp Met Glu Val 275 280 285Ser Cys Pro Asp Gly Tyr Thr Cys Cys Arg Leu Gln Ser Gly Ala Trp 290 295300 Gly Cys Cys Pro Phe Thr Gln Ala Val Cys Cys Glu Asp His Ile His 305310 315 320 Cys Cys Pro Ala Gly Phe Thr Cys Asp Thr Gln Lys Gly Thr CysGlu 325 330 335 Gln Gly Pro His Gln Val Pro Trp Met Glu Lys Ala Pro AlaHis Leu 340 345 350 Ser Leu Pro Asp Pro Gln Ala Leu Lys Arg Asp Val ProCys Asp Asn 355 360 365 Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cys GlnLeu Thr Ser Gly 370 375 380 Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala ValCys Cys Ser Asp His 385 390 395 400 Gln His Cys Cys Pro Gln Arg Tyr ThrCys Val Ala Glu Gly Gln Cys 405 410 415 Gln Arg Gly Ser Glu Ile Val AlaGly Leu Glu Lys Met Pro Ala Arg 420 425 430 Arg Gly Ser Leu Ser His ProArg Asp Ile Gly Cys Asp Gln His Thr 435 440 445 Ser Cys Pro Val Gly GlyThr Cys Cys Pro Ser Gln Gly Gly Ser Trp 450 455 460 Ala Cys Cys Gln LeuPro His Ala Val Cys Cys Glu Asp Arg Gln His 465 470 475 480 Cys Cys ProAla Gly Tyr Thr Cys Asn Val Lys Ala Arg Ser Cys Glu 485 490 495 Lys GluVal Val Ser Ala Gln Pro Ala Thr Phe Leu Ala Arg Ser Pro 500 505 510 HisVal Gly Val Lys Asp Val Glu Cys Gly Glu Gly His Phe Cys His 515 520 525Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln Gly Trp Ala Cys Cys 530 535540 Pro Tyr Ala Gln Gly Val Cys Cys Ala Asp Arg Arg His Cys Cys Pro 545550 555 560 Ala Gly Phe Arg Cys Ala Arg Arg Gly Thr Lys Cys Leu Arg ArgGlu 565 570 575 Ala Pro Arg Trp Asp Ala Pro Leu Arg Asp Pro Ala Leu ArgGln Leu 580 585 590 Leu

I claim:
 1. A method of inhibiting the growth or viability of hematopoietic malignant cells comprising contacting hematopoietic malignant cells with a GP88 antagonist wherein said antagonist inhibits the growth or viability of said hematopoietic malignant cells.
 2. The method of claim 1, wherein said hematopoietic malignant cells are leukemia cells of B cell lineage.
 3. The method of claim 1, wherein said hematopoietic malignant cells are multiple myeloma cells.
 4. The method of claim 1, wherein said GP88 antagonist is an anti-GP88 antibody.
 5. The method of claim 1, wherein said GP88 antagonist is a humanized anti-GP88 antibody.
 6. The method of claim 1, wherein said GP88 antagonist is a neutralizing anti-GP88 antibody.
 7. The method of claim 1, wherein said GP88 antagonist is an anti-GP88 antibody comprising a plurality of portions wherein at least one portion is derived from a human.
 8. The method of claim 1, wherein said GP88 antagonist is an anti-GP88 nucleic acid.
 9. The method of claim 8, wherein said anti-GP88 nucleic acid is selected from the group consisting of antisense nucleic acid and RNAi.
 10. The method of claim 8, wherein said GP88 antagonist inhibits the growth of said hematopoietic malignant cells by at least about 50%.
 11. The method of claim 8, wherein said GP88 antagonist inhibits the phophorylation activity of MAPK in said hematopoietic malignant cells.
 12. The method of claim 8, wherein said GP88 antagonist inhibits the activity of PI3 kinase in said hematopoictic malignant cells.
 13. A composition for inhibiting the growth of hematopoietic malignant cells, comprising a carrier and an GP88 antagonist in an amount sufficient to inhibit the growth of said hematopoietic malignant cells.
 14. The composition of claim 13, wherein said hematopoictic malignant cells are multiple myeloma cells.
 15. The composition of claim 13, wherein said GP88 antagonist is an anti-GP88 antibody.
 16. The composition of claim 13, wherein said GP88 antagonist is a neutralizing anti-GP88 antibody.
 17. A composition of claim 13, wherein said GP88 antagonist is an anti-GP88 antibody comprising a plurality of portions wherein at least one portion is derived from a human.
 18. A composition according to claim 13, wherein said GP88 antagonist is an anti-GP88 antibody capable of reducing the proliferation of lymphoma cells by at least about 50%.
 19. A composition according to claim 13, wherein said GP88 antagonist is an anti-GP88 nucleic acid.
 20. A composition according to claim 19 wherein said GP88 nucleic acid is selected from the group consisting of antisense nucleic acid and RNAi.
 21. A method for diagnosing B-cell leukemia comprising determining whether GP88 is present in a tissue sample containing B cells, wherein the presence of GP88 in said tissue sample indicates B-cell leukemia.
 22. The method of claim 21, wherein said B-cell leukemia is multiple myeloma.
 23. The method of claim 21, wherein said tissue comprises a material selected from the group consisting of blood, bone marrow, lymph, spleen, and liver.
 24. The method of claim 21, wherein said GP88 is GP88 protein.
 25. The method of claim 21, wherein said GP88 is GP88 nucleic acid.
 26. The method of claim 24, wherein said GP88 protein is detected with an anti-GP88 antibody.
 27. The method of claim 24, wherein said GP88 protein is detected with a humanized anti-GP88 antibody.
 28. The method of claim 24, wherein said GP88 protein is detected with an anti-GP88 antibody, wherein said anti-GP88 antibody is derived from an animal immunized with a material comprising the peptide of SEQ ID NO:
 3. 29. The method of claim 24, wherein said GP88 protein is detected with an anti-GP88 antibody, wherein said anti-GP88 antibody is derived from an animal immunized with a material comprising the peptide of SEQ ID NO:
 4. 30. The method of claim 24, wherein said GP88 protein is detected with an anti-GP88 antibody, wherein said anti-GP88 antibody is derived from an animal immunized with a material comprising the peptide of SEQ ID NO:
 5. 31. The method of claim 24, wherein said GP88 protein is detected with an anti-GP88 antibody, wherein said anti-GP88 antibody is derived from an animal immunized with a material comprising the peptide of SEQ ID NO:
 6. 32. The method of claim 24, wherein said GP88 protein is detected with an anti-GP88 antibody, wherein said anti-GP88 antibody is derived from an animal immunized with a material comprising the peptide of SEQ ID NO:
 7. 33. The method of claim 24, wherein said GP88 protein is detected by Western blot analysis.
 34. The method of claim 24, wherein said GP88 protein is by immunoassay.
 35. A method for diagnosing B-cell leukemia, comprising determining whether GP88 is present in a sample containing B-cells, wherein the presence of GP88 in said B-cells indicates B-cell leukemia.
 36. The method of claim 35, wherein said B-cell leukemia is multiple myeloma.
 37. A method for diagnosing multiple myeloma, comprising determining whether GP88 is present in bone marrow tissue, wherein the presence of GP88 in said bone marrow tissue indicates multiple myeloma.
 38. The method of claim 37, wherein the presence of GP88 in said bone marrow tissue is detected with an anti-GP88 antibody.
 39. The method of claim 37, wherein the presence of GP88 in said bone marrow tissue is detected by immunoassay.
 40. A method of determining whether a patient having a hematopoictic malignancy is responding or responsive to anti-cancer agents, comprising: measuring a first level GP88 in a tissue sample containing hematopoictic cells obtained from said patient at a first time; measuring the level of GP88 in a tissue sample containing hematopoietic cells obtained from said patient at a second time; comparing said first level of GP88 with said second level of GP88; and determining that a patient is not responding or responsive to anti-cancer agents if the second level of GP88 is higher than the first level of GP88.
 41. The method of claim 40, wherein said anti-cancer agent is a glucocorticoid or glucocorticoid analog.
 42. The method of claim 41, wherein said glucocorticoid or glucocorticoid analog is selected from the group consisting of dexamethasone, prednisolone, methylprednisolone, hydrocortisone, betamethasone, prednisone, fludrocortisone, cortisone, corticosterone, triamcinolone, and paramethasone.
 43. The method of claim 40, wherein said GP88 is detected with an anti-GP88 antibody or antibody fragment.
 44. The method of claim 40, wherein said GP88 is detected with an anti-GP88 nucleic acid.
 45. The method of claim 40, wherein said tissue sample is selected from group consisting of blood, bone marrow, lymph, spleen, and liver.
 46. The method of claim 40, wherein said hemapoietic cells are B-cell leukemia cells.
 47. The method of claim 40, wherein said hemapoietic cells are multiple mycloma cells.
 48. A method of treating hematopoietic malignancies with an anti-cancer agent in a patient comprising: determining a first level of GP88 present in a tissue sample containing hematopoietic cells obtained from said patient at a first time; determining a second level of GP88 present in a tissue sample containing hematopoietic cells obtained from said patient at a second time; comparing the first level of GP88 with the second level of GP88; and administering an anti-cancer agent to said patient in an amount sufficient to treat or prevent hemapoietic malignancies if the second level of GP88 is the same as or lower than the first level of GP88.
 49. The method of claim 48, wherein said anti-cancer agent is a glucocorticoid.
 50. The method of claim 48, wherein said anti-cancer agent is a glucocorticoid or glucocorticoid analog selected from the group consisting of dexamethasone, prednisolone, methylprednisolone, hydrocortisone, betamethasone, prednisone, fludrocortisone, cortisone, corticosterone, triamcinolone, and paramethasone.
 51. The method of claim 48, wherein said hematopoictic malignancy is B-cell leukemia.
 52. A method of determining whether a patient is responding or responsive to the anti-tumorigenic effects of anti-cancer therapy comprising: determining a first level of GP88 in hematopoeitic cells obtained from said patient at a first time; determining a second level of GP88 in hematopoeitic cells obtained from said patient at a second time; comparing said first level of GP88 with said second level of GP88; and determining that a patient is resistant to the anti-tumorigenic effects of anti-cancer therapy if the second level of GP88 is higher than the first level of GP88.
 53. The method of claim 52, wherein said anti-cancer therapy is glucocorticoid therapy.
 54. The method of claim 52, wherein said anti-cancer therapy comprises administering dexamethasone to a patient.
 55. A method of determining whether a patient is responding or responsive to the anti-tumorigenic effects of glucocorticoids or glucocorticoid analogs comprising: determining a first level of GP88 in a tissue sample containing B-cells obtained from said patient at a first time; determining a second level of GP88 in a tissue sample containing B-cells obtained from said patient at a second time; comparing the first level of GP88 with the second level of GP88; and determining that a patient is resistant to the anti-tumorigenic effects of glucocorticoids if the second level of GP88 is higher that the first level of GP88.
 56. The method of claim 55, wherein said GP88 is detected with an anti-GP88 antibody or antibody fragment.
 57. The method of claim 55, wherein said GP88 is detected with an anti-GP88 nucleic acid.
 58. The method of claim 55, wherein said glucocorticoids or glucocorticoid analogs are selected from the group consisting of dexamethasone, dexamethasone, prednisolone, methylprednisolone, hydrocortisone, betamethasone, prednisone, fludrocortisone, cortisone, corticosterone, triamcinolone, and paramethasone.
 59. The method of claim 55, wherein said tissue sample is selected from the group consisting of blood, bone marrow, lymph node, spleen, and liver. 